Protein-proteophore complexes

ABSTRACT

The application relates to a composition comprising a hyperbranched polymer attached to a core and a biologically active moiety. The biologically active moiety is attached to the core by means of a substantially non-enzymatically cleavable linker L. The composition can be used to deliver the biologically active moiety to its target.

The present invention relates to inclusion compounds comprising aprotein and an encapsulating organic compound (EOC), which will also bereferred to as proteophore, in a 1:1 stoichiometrie. In one embodiment,the EOC is a dendrimer, resulting in a dendrimer-protein inclusioncompound (DPIC). In a further embodiment, the EOC is a macrocyclicstructure resulting in a macrocycle-protein inclusion compound (MPIC).The encapsulating compounds, as well as the resulting inclusioncompounds, are water soluble and lend themselves for the controlledrelease of the protein to a target, preferably in a living body, inparticular a mammal.

Proteins are large and unstable molecules. A large amount of proteins isknown which show an important pharmacological activity. Examples includeinsulin, interferon, growth hormones and blood forming factors. Ingeneral, proteins are applied to mammals and humans by injection. Todate, it is not possible to apply pharmacologically active proteinsorally or transdermally. Following the injection, the proteins arereadily attacked and often partially or totally eliminated by the immunesystem, various enzymes or kidney filtration. In addition, the proteincan be toxic or cause allergic reactions.

In order to overcome the in-vivo elimination, several techniques weredeveloped in order to ensure a controlled release of the respectiveprotein. Examples include modification of the protein sequence,pegylation, proteinylation, binding to albumin, glycosylation,formulation into hydrogels or encapsulation by microparticles,nanoparticles, dendrimers.

Supramolecular chemistry is directed towards the synthesis and analysisof inclusion compounds in which two or more components are associatedthrough complete enclosure of one set of molecules in a suitablestructure formed by another.

In such chemical host-guest systems, one molecular entity iscomplementary to a different, second entity. Complementarity can occurin shape or physicochemical properties or in a combination of both. Inthe case of shape complementarity, the host molecule forms a cavity ofsize similar to the guest molecule. In such topologically well-definedcases, where the cavity is an inherent structural feature of a singlemolecule, the host is termed cavitand, and the host-guest aggregatecavitate. Typically, the stoichiometry of the supramolecular system is1:1. Nevertheless, various types of complex stoichiometries are known.Guest molecules are typically smaller in size than the correspondinghost compound.

Examples for small host-guest systems are for instance complexes formedbetween crown ethers and sodium or potassium ions. Well-known examplesfor synthetic organic host-guest molecules are the complexation ofaromatic compounds such as nitrophenol by cyclodextrin carbohydrates.Cyclodextrins come in various ring sizes, the larger of which canaccomodate bicyclic structures such as naphthalene and derivatives.Fullerene molecules are of spherical shape and accomodate free space ofa diameter of 0.7 nm.

Biomacromolecules such as starch can form inclusion complexes with smallguests such as iodine by filling a channel-like volume in the interiorof a helix. DNA double helices are known to accomodate rigid aromaticcompounds by means of intercalation. Serum albumin is a well-studiedexample of a protein that can be loaded with several molecules of fattyacids.

Comparatively little research has been undertaken to investigate thecomplexation of biomacromolecules such as proteins by synthetic hostcompounds.

A large number of molecular medicines are based on proteins or peptidesof which group insulin, interferons, growth hormones and blood factorsare among the most widely used therapeutics.

Protein therapeutics are known to suffer from various drawbacks.Proteins are inherently instable macromolecules as their bioactivitydepends upon the correct three-dimensional positioning of itspolypeptide chain. External factors such as solvents, surfaces,agitation, temperature or pH may effect the conformational equilibriumand result in partial or total unfolding, denaturation, agglomeration orprecipitation. Proteins of non-human origin or proteins containingnon-human amino acid sequences are highly immunogenic. Antibodyformation is even notable for human proteins such as insulin iffrequently administered by injection. Biomolecules may be cleared fromcirculation too fast or too slow for a given therapeutic application andmay exhibit a narrow therapeutic window. Proteins can be degraded byendogenous proteases. Most therapeutic biomolecules need to beadministered parenterally, often imposing the need for lifetime dailyinjections on the patient. At this point in time there are no approvedprotein formulations for oral or pulmonary delivery. Only in a few caseshas it been possible to direct the therapeutic protein to the diseasedtissue or cell type or to deliver the protein in an intracellularfashion.

Therefore it is highly desirable to develop molecularly defined deliveryvehicles to enhance the therapeutic benefit of protein-based medication.Specifically, proteins need to be efficiently encapsulated forprotection and released from the encapsulating agent for bioactivity.

Proteins are composed of condensated amino acid sequences that fold intoa compact three-dimensional arrangement, often of globular shape. Thediameter for globular proteins typicaly ranges from 1 to 10 nm. Proteinscan form complexes comprised of several identical or different subunits,and several proteins can associate to form even larger complexes. Aprotein encapsulating agent has to provide a well-hydrated internalvolume of a similar size and approximately spherical or ellipsoidal orchannel-like shape. The water content is an important molecularproperty, as most biomolecules depend on a hydration shell forbioactivity.

Polymers have proven highly useful in delivery of therapeutic molecularbiological material to humans. Linear or branched water soluble polymerscan occupy a volume of similar size or greater than a protein molecule.

Polyethyleneglycol (PEG) is a polymer of low toxicitiy andimmunogenicity. Various therapeutic proteins have been covalentlyconjugated to PEG by a process called PEGylation and successfullyapplied in molecular therapy (Harris J M, Chess R B, Nature (2003)214-21).

An advantage of protein PEGylation is the improvement of pharmacokineticproperties of the conjugate in circulation. Unconjugated proteins suchas interferon alfa-2a are cleared rapidly within 2.5 h in rats, thecorresponding PEGylated interferons circulate with a half-life of t½=3.4h (linear 5 kDa PEG monoconjugate) up to 23 h (dipegylated with 2×20 kDaPEG). This effect is attributed to reduced renal filtration. Kidneyfiltration is partially a size-exclusion process, and enhancing thehydrodynamic radius of a protein by PEGylation can significantly reduceits rate of clearance. PEG is strongly hydrated (2-3 water molecules perethyleneoxide unit) and therefore displays a high apparent molecularweight in size exclusion chromatography studies. Due to its highconformational flexibility and hydration, PEG molecules appear 5-10times as large as proteins of similar molecular mass.

PEG is widely used to render surfaces protein adsorption resistant andto precipitate proteins from aqueous solution, corroborating the notionthat PEG does not physicochemically bind to protein.

The property of PEG to fold randomly and to occupy a large molecularvolume explains also for the second therapeutic benefit of PEGylation,namely the reduction of immunogenicity of proteins. This effect is mostpronounced for proteins of non-human origin and is likely to be achievedby imposing a steric shield in the vicinity of the immunogenic epitopeand thereby preventing recognition by the immune system.

The shielding effect may be enhanced by employing branched PEG.Polyethylene glycol with a low degree of branching is known from U.S.Pat. No. 5,643,575 and the 2003 catalogue of Nektar Therapeutics. WO01/21197 mentions branched monosubstituted insulin-PEG conjugates.

The steric shielding mechanism explains for the observed reduction ofbioactivity of PEGylated proteins. Covalent conjugation of protein sidechains close to an epitope generally may impair the ability of theprotein to bind to its receptor. Care has to be taken to identify areactive protein side chain that is in a distal position to the regionof the protein surface that is mediating receptor binding or enzymaticactivity. For this reason, PEG monoconjugation is preferred overmultiple conjugation. Nevertheless even for monoconjugates, variousregioisomers are obtained in various ratios.

Steric shielding may also be enhanced by conjugating the protein to morethan one PEG molecule. Multiple PEGylation leads to an apparent increasein hydrodynamic volume and serves better to protect the protein fromantibody recognition or protease attack. The approach is compromised byloss of bioactivity, loss of therapeutic activity per gram of materialand by increasing the risk of protein inactivation by conformationaldestabilization.

It is a challenge to obtain a homogeneous product from the reaction ofprotein with PEG reagent. If a PEG reagent is reacted with a givenprotein under equimolecular conditions or added in slight excess, mono-,bis-, tris- and oligo-conjugations are commonly obtained. The reason isthat protein surfaces display various functional groups of similarreactivities. The difficulties in analysis of such mixtures isaggravated by the fact that PEG in itself is a polydisperse molecule.Polydispersity relates to the fact that PEG cannot be obtained as amolecule of precise chain length beyond a degree of polymerization (dp)of 12 ethyleneglycol units. Typical PEGs of MW 5 kDa or 20 kDa exhibitpolydispersities of 1.01 up to 1.20 respectively.

Non-water soluble polymers such as poly(lactide-co-glycolide, PLG) mayform nano- or microparticles if precipitated from aqueous solution undercertain conditions. The formed particles are not water-soluble but aresuspended in aqueous solution. Proteins present in the aqueous phase maybe entrapped inside these non-covalent assemblies. Proteins are releasedas the particles degrade. Such hydrogels are successfully used in slowrelease formulations of therapeutic proteins such as growth hormone(Tracy M A, Biotechnol Prod 14 (1998) 108-15).

Proteins or polypeptides may be incorporated in polymeric material bycarrying out the polymerization step in the presence of the biomolecule.Insulin has been loaded to n-butylcyanoacrylate nanoparticles in thisfashion (WO 96/31231). Polymerizing monomers are highly reactivemolecular species and the process usually requires organic solvents.Biomacromolecules may suffer structural modification or degradationunder such conditions.

Typical encapsulation methods involving prepolymerized entities arewater-in-oil-in-water (w/o/w) double emulsion/solvent evaporation or thesolid-in-oil-in-water (s/o/w) technique. The encapsulation processinvolves organic solvents such as methylene chloride, heat andsonication or homogenization and therefore can lead to inactivation ofthe encapsulated material.

An alternative method is based on polymer crosslinking. Proteins may bepermanently entrapped in polymers if the crosslinking step is carriedout in the presence of the protein. Protein, monomers and crosslinkerare mixed and polymerized. Such polymers are not soluble per se.Crosslinked polymers are constituted of a network of polymer chains.Within this network various pores and cavities and channels exist in arandom fashion, some of which may be sufficiently large to allow fordiffusion of protein into, through or out of the polymer. The degree ofcrosslinking has a strong effect on diffusion into and effusion from thepolymer. Products from such crosslinking are called hydrogels, as theycan be produced from water-soluble, well-hydrated components and exhibitconsiderable swelling behaviour.

All of these methods of preparation may have severely detrimentaleffects on the protein integrity and bioactivity. As a portion of theprotein material is inactivated during the particle preparation process,and it is difficult to quantify the remaining bioactivity of theentrapped protein sample.

Even after encapsulation, the protein is put under stress as the proteinis forced to make tight contact with the more hydrophobic polymermolecules. This again may cause additional denaturing and loss ofactivity. Additionally, the molecular architecture of the polymernetwork imposes mechanical and physicochemical stress on the protein.The protein may be dehydrated or denatured by aggregation or contact tointernal surfaces, and it is difficult to analyze the protein'sbioactivity after encapsulation.

The release of proteins from the entrapment can be achieved bydiffusion, a chemical or enzymatic reaction leading to degradation ofthe polymer or solvent activation (through osmosis or swelling) or acombination of mechanisms. For therapeutic applications, effusion,swelling or biodegradation mechanisms take place in vivo and aredifficult to control.

Liposomes can form small unilamellar vesicles or large, multilamellarassemblies (Refs). The encapsulation of drugs in liposomes has beenstudied extensively and is applied in molecular therapy. WO 03/030829describes liposome-encapsulated insulin formulations. Typical techniquessuch as mixing the drug with the lipid in an organic solvent, additionof an aqueous medium and subsequent removal of the organic solvent ordialysis of mixed lipid-detergent micelles are not readily applied toprotein encapsulation due to protein denaturation by solvent ordetergent. A more suitable approach is lipid film hydration. Liposomesare formed by hydrating and dispensing a previously dried film of lipid.Liposomes are not per se water-soluble but can be homogeneouslydistributed in water by means of dispersion. If protein is present inthe hydration solution it becomes both associated on the surface andentrapped in the interior of the liposomes. The process reduces theexposure of protein to denaturing conditions but is of littleencapsulation efficiency.

Dendrimers are well-defined polymeric structures. Dendrimers are basedon repeating hyperbranched structures emanating from a central core(U.S. Pat. No. 4,507,466). Typical dendrimers are based onpolyamidoamine (PAMAM), polyethylene imine (PEI), polypropylene imine orpolylysine. These synthetic macromolecules are assembled in a stepwisefashion, with each reaction cycle adding another layer of branches(dubbed “generation”). Dendrimers are synthetically accessed bystepwise, divergent “bottom-up” or convergent “top-down” synthesis.Central structural component is the core unit from which hyperbrancheddendrimers extend in a radially symmetric fashion. The core may provideat least two reactive groups for dendrimer conjugation, it may also beof heterofunctional nature and protecting groups may be used. In thelatter case, the dendrimer may be assembled, and a guest compound may besubsequently conjugated to an anilin core by means of orthogonalchemistries (WO 88/01180). The core and dendrimers form the interior orbackbone of a dendrimer. As a consquence of the spherical symmetrysupported by sterical crowding, the terminal groups of the hyperbranchesare defining the exterior. In higher generation dendrimers, the terminalbranches form rather dense shells and flexible internal voids have beendiscovered. It is understood, that for a given dendrimer these cavitiesare filled up by backfolded end groups and tightly coordinated solventmolecules. Dendrimers are related to micelles, similary well suited tocomplex hydrophobic compounds. But in contrast they exhibit higherstructural order because of their monomolecular nature and the absenceof a dynamic equilibrium of various species. Synthetic compounds canonly diffuse into dendrimers if certain structural requirement such asconformational rigidity and flatness as well as charge distribution suchas affinity to tertiary amines are met. Various apolar compounds such aspyrene or naphthalene have been encapsulated in dendrimers, but thenumber of trapped guests as well as their molecular interaction with thedendrimer interior are rater undefined and frequently substoichiometric.

In U.S. Pat. No. 5,714,166 and WO 95/24221, dendrimer-protein conjugatesare revealed. PAMAM dendrimers of G4 are covalently coupled throughtheir terminal functional groups to insulin, fluorescently labeledinsulin, avidin, monoclonal antibodies and bradykinin. The reactivegroups used for conjugation are only present at the surface of thedendrimers, and therefore any covalent adduct generated by the teachedmethod will be associated with the dendrimer exterior. Sterical“congestion” of the dendrimeric terminal groups is a prerequisite to theformation of internal void space. In a scanning transmission electronmicrograph study, it was observed that PAMAM dendrimers undergo amorphological change at the G9 stage. Surface congestion created ahollow interior surrounded by a dense rim. The G4 dendrimers used forprotein conjugation do not contain such voids. Furthermore it isapparent from molecular size comparison, that a 3 nm sized insulin maynot be encapsulated in a dense, 4 nm-sized generation 4 PAMAM dendrimer.Hemoglobin has a diameter of 5.5 nm, and PAMAM dendrimers of G5, G6 andG7 exhibit diamters of 5.3 nm, 6.7 and 8.0 nm respectively.Macromolecules such as peptides and proteins are per se excluded fromdiffusion through the dense molecular packing and entering the interiorof such dendrimers. As the dendrimer surface is rather denselyclustered, pore sizes are too small to allow for an entry of a proteininto the dendrimer interior. For these reasons, macromolecular proteinor polypeptide guests have not been encapsulated in dendrimers, neitherhas the non-covalent encapsulation of proteins been demonstrated.

PAMAM dendrimers contain free amine groups on their surfaces and readilyassociate with DNA through electrostatic interactions.

WO 01/07469 details water-soluble polypeptide dendrimers constituted ofornithin and glycine amino acids. The patent application also teachesthe non-covalent encapsulation of an oligosaccharide, heparin, bydendrimerization of the dendrimer core in presence of heparin under mildconditions. The oligosaccharide is released from the dendrimer bylight-induced cleavage of UV-labile bonds within the dendritic backbone.The core structure used here was tris(2-maleimidoethyl)amine.Presynthesized polypeptide dendrimers, containing a free thiol groupwere incubated in DMF in the presence of heparin. This approach isunlikely to be applicable to proteins as substantial side reactionsbetween the maleimido core and the protein will occur, furthermoresteric competition will prevent an efficient encapsulation as eitherfull formation of the tri-dendritic structure is prevented or theprotein will not be entrapped. The example does not teach how togenerate a complex of well-defined stoichiometry.

There is a continuous need for techniques and devices which allow for aneffective encapsulation of proteins in order to ensure a controlleddelivery and, if appropriate, release of pharmacologically activeproteins. The encapsulation should not alter the proteins' structure andproperties and should efficiently protect the protein from attacks bythe immune system and enzymes of the individual to which the protein isadministered. Furthermore, the protein should enable an efficientrelease of the encapsulated protein, in case this is desired.

This object is attained by a protein encapsulated covalently ornon-covalently by an encapsulating organic compound (EOC) wherein theprotein and the encapsulating organic compound are present in 1:1stoichiometry.

Appropriate EOCs are water soluble.

The EOCs contain several, i.e. at least 2, molecule chains of anappropriate length which chains can arrange such that a cavity is formedwhich can accommodate the protein and protect it from the action ofenzymes, antibodies and the like. The molecule chains will hereinafterbe referred to as “encapsulating molecular chains” EMC. The EMCs can bedirectly connected with each other, or via a chemical unit, often one ormore so-called branching units (see below).

In the following, EMCs according to the present invention will bedefined. This definition applies every time EMCs will be mentioned inthe present application in a general form, either in connection wit ageneral formula or in any other context.

The EMCs contain hydrophilic groups, in an appropriate ratio and amountwith respect to hydrophobic groups which may be present in the EOC, torender the latter water soluble.

The EMCs are built up from linear, branched or cyclical alkyl chains. Torender the hydrophobic alkyl chains more hydrophilic, hetero atoms likebut not limited to S, N, O may be present within the chain. Furtherappropriate groups which can be present in the EMCs include (—S—S)—,amide —C(O)NH— or C(O)NR—, —S-succinimido, amino (—NR—), carboxylicester (—C(O)O—), sulfonamide (—S(O)₂—NR—, carbamate (—O—C(O)—NR—),carbonate (—O—C(O)—O—), sulfone (—S(O)₂—), ether (—O—), oxime(—CR═N—O—), hydrazone (—CR═N—NR—), urea (—NR—C(O)—NR—), thiourea(—NR—C(S)—NR—), carbohydrate, glyceryl, phosphate (—O—P(O)(OR)O—),phosphonate (—P(O)(OR)O—), saturated and nonsaturated (hetero)cycliccompounds. Non-limiting examples of R include H, linear, branched orcyclical alkyl groups which may contain further functional groups orhetero atoms. In addition to the afore-mentioned groups, further groupsknown to the person skilled in the art can be present in the EMCs.

Example for preferred groups in the EMCs comprise oxyalkylene groups(i.e. oxyethylene (—OCH₂CH₂)—, oxypropylene groups (—OCH₂CH(CH₃))— andoxybutylene groups) and amide groups (—C(O)NH)—. It is preferred if theEMCs comprise oxyethylene groups (—OCH₂CH₂)— and amide groups(—C(O)NH)—.

In one embodiment of the present invention, the EMCs comprises at leastone amino acid unit in its chain. In the context of the presentinvention, “amino acid unit” means an amino acid, preferably a naturallyoccurring amino acid like lysine, which is connected to at least onefurther binding partner, for example a further amino acid, by its aminoand/or its carboxy function. The amino acid may be modified, e.g. carryone or more substituents.

The EMCs can carry one or more substituents (capping groups or modifiersC) on their backbone. Appropiate capping groups are sterically demandinggroups. The capping groups will in particular be present if the EMCsrequire sterically demanding groups forcing them into a certainconformation necessary for the creation of the cavity enclosing theprotein. In many cases, the EMCs according to the present invention arenot rigid, and the subunits of the EMCs may rotate around the bonds ofthe chain and occupy a spatial position in accordance with the stericalrequirements (which, in general, will be the position with the lowestenergy). The capping groups can avoid a too close approaching of theEMCs and an opening of the cavity which may result in an insufficientencapsulating of the protein and an insufficient protection from theattack of enzymes, antibodies or the like. Furthermore, the protein maytotally leave the cavity through the gap resulting from the movement ofthe EMCs.

The capping groups C are built up from linear, branched or cyclicalalkyl chains. To render the hydrophobic alkyl chains more hydrophilic,hetero atoms like but not limited to S, N, O may be present within thechain. Further appropriate groups which can be present in the cappinggroups include (—S—S)—, amide —C(O)NH— or C(O)NR—, —S-succinimido, amino(—NR—), carboxylic ester (—C(O)O—), sulfonamide (—S(O)₂—NR—, carbamate(—O—C(O)—NR—), carbonate (—O—C(O)—O—), sulfone (—S(O)₂—), ether (—O—),oxime (—CR═N—O—), hydrazone (—CR═N—NR—), urea (—NR—C(O)—NR—), thiourea(—NR—C(S)—NR—), carbohydrate, glyceryl, phosphate (—O—P(O)(OR)O—),phosphonate (—P(O)(OR)O—), saturated and nonsaturated (hetero)cycliccompounds. Non-limiting examples of R include H, linear, branched orcyclical alkyl groups which may contain further functional groups orhetero atoms. In addition to the afore-mentioned groups, further groupsknown to the person skilled in the art can be present in the EMCs.

In one embodiment of the present invention, the capping units compriseat least one amino acid unit in its chain. In the context of the presentinvention, “amino acid unit” means an amino acid, preferably a naturallyoccurring amino acid like lysine, which is connected to at least onefurther binding partner, for example a further amino acid, by its aminoand/or its carboxy function. The amino acid may be modified, e.g. carryone or more substituents.

It is preferred if the capping groups comprise oxyalkylene groups (i.e.oxyethylene (—OCH₂CH₂)—, oxypropylene groups (—OCH₂CH(CH₃))— andoxybutylene groups) and amide groups (—C(O)NH)—. It is even morepreferred if the capping groups comprise oxyethylene groups (—OCH₂CH₂)—and amide groups (—C(O)NH)—, in an appropriate ratio and amount, inorder to obtain capping groups with the desired hydrophilicity which maybe higher or lower than the hydrophilicity of the EMCs.

The capping groups in the EMC can contain one or more functional groupsfrom those cited above. The functional groups present in a given cappinggroup can be identical or different. Each of the cited groups can bepresent only once or several times. The capping groups present in agiven EMC can be identical or different.

In one embodiment of the present invention, the capping groups do nothave a high branching degree. This will in particular be the case if theEOCs according to the present invention have a high number of EMCs.

In a further embodiment of the present invention, the capping groups arehighly branched molecules having preferably a branching degree of 2, 3,4, 5 or 6. A branching degree of 2 means that the principal chainconnected to the encapsulating unit splits up into 2 subchains, whereasin the case of a branching degree of 3, the main chain splits up into 3subchains, etc.

The subchains may themselves also be branched. In the context of thepresent invention, this case will be referred to as “subbranched” (Themodifiers are subbranched, i.e. their main chain contains subchainswhich themselves are branched.) For example, in the case of a branchingdegree of 2, each of the 2 subchains can be subbranched to asubbranching degree of 2, meaning that also each of the 2 subchains intowhich the main chain (principal chain) splits up itself splits up into 2subchains. In such case, the branching degree will be designated as2(2).

When an EOC according to the present invention is substituted by cappinggroups which are highly branched, a dendritic structure will result.

The term hyperbranched polymer used in this description is intended toinclude a combination of an EMC with capping groups as well as EOCs.

In one embodiment of the present invention, the encapsulation isrealized by an EOC-protein inclusion compound EPIC according to theformula (I) in which the EMCs are connected to each other by onebranching unit B, resulting in an EOC of the structure according to theformula (II) in which a cavity is formed.

In the formulae (I) and (II), the symbols have the following meanings:

-   -   B: branching unit (basic unit, core) containing at least one        branching center Bc and at least two branching functional groups        Bfg connected to or capable of reacting with an encapsulating        unit EMC;    -   EMC: encapsulating molecular chain;    -   L: linker containing at least one functional group Lfg which is        connected to the protein P or capable of connecting with        functional groups present on the protein P under the formation        of a chemical bond;    -   l: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, preferably 3, 4, 5,        6, 7 or 8, in particular 2,3,4,5 or 6;    -   P: pharmacologically active protein.

The EMCs have been defined beforehand. In the following, the groups B, Land P according to the formulae (I) and (II) will be defined. Thisdefinition applies every time B, L and P will be mentioned in thepresent application in a general form, either in connection with ageneral formula or in any other context.

The EOCs according to the present invention may comprise more than 2EMCs, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13. In theformulae below, some preferred embodiments are shown.

The branching units B can be regarded as the basic unit or core of theEMCs according to the present invention.

The EMCs are linked by an EMC functional group to at least one branchingunit B. B contains at least one branching center Bc. Examples of Bcinclude units like >CH—or >C< and the respective analogues wherein H isreplaced by an organic group; >N—; >P—. The centers Bc can directly belinked to the branching functional groups (see below), or can be linkedto at least one organic chain.

Examples for appropriate organic chains include linear, branched orcyclical alkyl chains. Hetero atoms like but not limited to S, N, O maybe present within the chain. Further appropriate groups which can bepresent in B include (—S—S)—, amide —C(O)NH— or C(O)NR—, -S-succinimido,amino (—NR—), carboxylic ester (—C(O)O—), sulfonamide (—S(O)₂—NR—,carbamate (—O—C(O)—NR—), carbonate (—O—C(O)—O—), sulfone (—S(O)₂—),ether (—O—), oxime (—CR═N—O—), hydrazone (—CR═N—NR—), urea(—NR—C(O)—NR—), thiourea (—NR—C(S)—NR—), carbohydrate, glyceryl,phosphate (—O—P(O)(OR)O—), phosphonate (—P(O)(OR)O—), saturated andnonsaturated (hetero)cyclic compounds. Non-limiting examples of Rinclude H, linear, branched or cyclical alkyl groups which may containfurther functional groups or hetero atoms. In addition to theafore-mentioned groups, further groups known to the person skilled inthe art can be present in B.

B can contain one or more groups chosen from those cited above. Thegroups can be identical or different. Each of the cited groups can bepresent only once or several times. In a preferred embodiment of thepresent invention, B comprises at least one amino acid unit, preferablyof a naturally occurring amino acid like lysine. It is even morepreferred if B contains a unit composed of several amino acid units.

In general, B will be a branched structure containing one or more of theabove mentioned groups and having a certain length, in accordance withthe steric requirements of the protein to be encapsulated. B willcomprise one or more branching center. Furthermore, B will contain atleast two branching functional groups Bfg allowing for the attachment ofthe EMCs.

Examples for suitable bond species formed between B and the EMC includethe following: (—S—S)—, S-succinimido, amide —C(O)NH— or C(O)NR—,—S-succinimido, amino (—NR—), carboxylic ester (—C(O)O—), sulfonamide(—S(O)₂—NR—), carbamate (—O—C(O)—NR—), carbonate (—O—C(O)—O—), ether(—O—), oxime (—CR═N—O—), hydrazone (—CR═N—NR—), urea (—NR—C(O)—NR—),thiourea (—NR—C(S)—NR—), phosphate (—O—P(O)(OR)O—), phosphonate(—P(O)(OR)O—).

Non-limiting examples of R include H, linear, branched or cyclical alkylgroups which may contain further functional groups or hetero atoms oraryl groups.

As will be apparent to the person skilled in the art from the foregoing,the functional groups present on the EMC and B (Bfg) of the EOC will betransformed (and changed) into a bond species. When, in the context ofthe present invention, reference is made to “functional group” presenton B and the EMC, this refers to the functional groups as such, as wellas to the bond species formed by reaction between the groups.

In the context of the present invention, “bond” or “chemical bond”refers to the attraction forces as such between two or more atoms (e.g.a “covalent bond”), whereas “bond species” denotes the chemical bond andthe atoms in the vicinity which is involved in the binding process (e.g.—S—S—).

As the bond species depicted beforehand are formed by the reactionbetween functional groups (which functional groups can be identical ordifferent), the EMCs and B before forming the bond species, both containfunctional groups which are capable of reacting with each other underthe formation of an appropriate chemical bond, preferably one of thebonds mentioned beforehand.

Examples for appropriate branching functional groups Bfg and EMCfunctional groups comprise amino (—NRH), carboxylic acid (—C(O)OH) andderivatives, sulfonic acid (—S(O)₂—OH) and derivatives, carbonate(—O—C(O)—O—) and derivativs, hydroxyl (—OH), aldehyde (—CHO), ketone(—CRO), hydrazine (H₂N—NR—), isocyanate (—NCO), isothiocyanate (—NCS),phosphoric acid (—O—P(O)(OR)OH) and derivatives, phosphonic acid(—P(O)(OR)OH) and derivatives, haloacetyl, alkyl halides, maleimide,acryloyl, arylating agents like aryl fluorides, hydroxylamine,disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone,diazoalkanes, diazoacetyl compounds, epoxide, oxirane, aziridine,Non-limiting examples of R include H, linear, branched or cyclical alkylgroups which may contain further functional groups or hetero atoms oraryl groups.

In a preferred embodiment of the present invention branching functionalgroups and EMC functional groups comprise amino, carboxylic acid andderivatives, hydrazine, hydroxylamine, thiol, aldehyde, hydroxyl,carbonate, maleimide or haloacetyl groups.

As already mentioned, B can contain two or more Bfgs. This is inparticular the case when the EOC comprises more than four, for example5, 6, 7, 8 or more EMCs.

Likewise B can contain one or more branching centers Bc.

A further essential constituent of the EOCs according to the presentinvention is the linker L which serves to establish a chemical bondbetween the protein P and the EOC, by the reaction between appropriatefunctional groups Lfg on the linker L and the protein. The chemical bondcan be a covalent bond or a non-covalent bond, for example acoordinative bond. Preferably, the EOC has 1 or 2 linkers.

In the context of the present invention, several sorts of linkers can beemployed:

Non-cleavable linker: a linker containing no selectively cleavablebonds.

Cleavable linker: a linker containing a bond that can be selectivelycleaved by a cleavage reagent (TCEP, TFA, DTT, enzyme, or a buffer).

Traceless linker: a linker that upon cleavage releases protein in such afashion that the protein is not associated with a remaining linkercleavage product.

Prodrug linker: a cleavable linker containing a bond that can beselectively cleaved under in-vivo conditions, for instance in thepresence of endogeneous enzymes or other endogeneous reagents, or solelyin aqueous buffer.

Traceless prodrug linkers: linkers having both the characteristics ofprodrug linkers and traceless linkers.

Depending on the therapeutic application, the protein may need to bepermanently encapsulated, and therefore non-cleavable stable linkers maybe employed. This is exemplified in the preparation of hemoglobin-EOCconjugates. Hemoglobin requires the diffusion of oxygen through the EOCbut the protein does not need to be released from its encapsulation forbioactivity. Corresponding linkers are known in the art (Hermanson G T,Bioconjugate Techniques, Academic Press San Diego, 1996).

In many cases, the release of the protein from the EOC/EPIC is mandatoryfor its bioactivity. One example is insulin which, in order to bind toits receptor, must diffuse out of the shielding EOC. Protein release maybe achieved by cleaving the covalent tether between protein and EOC.

Cleavable linkers are known in the art (see Hermanson).

The linker L can react with any appropriate functional group Pfg presenton the protein P, preferably with those mentioned below.

Examples for suitable bond species formed between the protein P and thelinker L include the following: (—S—S)—, S-succinimido, amide —C(O)NH—or C(O)NR—, —S-succinimido, amino (—NR—), carboxylic ester (—C(O)O—),sulfonamide (—S(O)₂—NR—), carbamate (—O—C(O)—NR—), carbonate(—O—C(O)—O—), ether (—O—), oxime (—CR═N—O—), hydrazone (—CR═N—NR—), urea(—NR—C(O)—NR—), thiourea (—NR—C(S)—NR—), phosphate (—O—P(O)(OR)O—),phosphonate (—P(O)(OR)O—). Non-limiting examples of R include H, linear,branched or cyclical alkyl groups which may contain further functionalgroups or hetero atoms or aryl groups.

In the context of the present invention, “bond” or “chemical bond”refers to the attraction forces as such between two or more atoms (e.g.a “covalent bond”), whereas “bond species” denotes the chemical bond andthe atoms in the vicinity which is involved in the binding process (e.g.—S—S—).

As the bond species depicted beforehand are formed by the reactionbetween functional groups (which functional groups can be identical ordifferent), the EOC, before reacting with the protein, and the protein,before reacting with the EOC, both contain functional groups which arecapable of reacting with each other under the formation of anappropriate chemical bond, preferably one of the bonds mentionedbeforehand.

Examples for appropriate protein functional groups Pfg which are part ofthe amino acids forming the natural (i.e. non-modified) protein areamino, thiol, hydroxyl, phenol, imidazole, amide, indole, carboxylicacid and guanidino groups.

In a preferred embodiment of the present invention Pfgs comprise amino,imidazole and thiol groups.

Examples for appropriate linker functional groups comprise amino (—NRH),carboxylic acid (—C(O)OH) and derivatives, sulfonic acid (—S(O)₂—OH) andderivatives, carbonate (—O—C(O)—O—) and derivatives, hydroxyl (—OH),aldehyde (—CHO), ketone (—CRO), isocyanate (—NCO), isothiocyanate(—NCS), haloacetyl, alkyl halides, maleimide, acryloyl, arylating agentslike aryl fluorides, disulfides like pyridyl disulfide, vinyl sulfone,vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide, oxirane,aziridine, Non-limiting examples of R include H, linear, branched orcyclical alkyl groups which may contain further functional groups orhetero atoms or aryl groups.

In a preferred embodiment of the present invention Lfgs comprisecarbamate, carbonate, thiol, thioether, succinimidyl, amide anddisulfide.

As will be apparent to the person skilled in the art from the foregoing,the functional groups present on the protein P (Pfg) and the linker L(Lfg) of the EOC will be transformed (and changed) into a chemical bond.When, in the context of the present invention, reference is made to“functional group” present on the linker L and the protein P, thisrefers to the functional groups as such, as well as to the bond speciesformed by reaction between the groups.

A part of the present invention are traceless double prodrug linkerstructures and their EPICs resulting in a novel mechanism of cleavageand subsequent release of the protein from the EOC.

Many widely applied and commercially available protein linker reagentscleave in such a fashion, that part of the linker remains conjugated tothe protein. As such linker fragments are of low molecular weight and ifthe site of conjugation does not involve an amino acid that is essentialfor receptor binding, the bioactivity of the therapeutic protein may befully or partially retained.

More advantageous are cleavable, traceless linkers that release theprotein in an unmodified form under in vivo conditions such as neutralpH without the addition of chemical or biological cleaving agents.Examples are double prodrugs which are based on linker moieties whichare cleaved in a two-step process in vivo. WO 99/30727A1, which isincorporated herein by reference, reveals conjugates containing a PEGmoiety, a double prodrug linker and protein. The advantage of suchsystems is that the protein is released in an unmodified form. Thelinker cleavage process is traceless, the protein end product of thecleavage step do not contain remnants of the linker structure. In afirst, rate-determining step one bond is hydrolyzed. This is typicallyan ester bond, such as in a phenol ester, and hydrolysis may occur byenzymatic attack (lipases) or autohydrolysis or a combination of both.The resulting free phenol is instable and rapidly rearranges forinstance through 1,4- or 1,6-arylelimination, and cleavage of acarbamate releases the protein, CO₂ and an instable aromatic moiety.

Furthermore, linker are known to the person skilled in the art that canbe cleaved in such a fashion that after cleavage no parts of the linkerremain at the EOC.

Thus, a preferred embodiment of the present invention are tracelessprodrug linkers which contain an ester functionality, in particular aphenol ester functionality, and a carbamate functionality.

Examples for suitable linker reagents are those according to theformulae (1), (2), (5), (6) and (7).

The most preferred linker reagent is the linker reagent according to theformula (11) below (traceless prodrug linker).

Lee S, Greenwald R B, McGuire J, Yang K, Shi C, Bioconjugate Chem 12(2001) 163-9, which is incorporated herein by reference, reviewed doubleprodrug linkers employing 1,6-elimination for releasable PEG-proteinconjugates. The use of such or related linkers for EOC-proteinconjugates is within the scope of the present invention.

After the cleavage of the linker, a EPIC results in which the protein isnot connected to the EOC via a linker (a chemical bond), but the proteinis held within the cavity defined by the EOC. The resulting EPICs are anobject of the present invention. It depends on the release kinetics ofthe protein if the respective EPIC having no bond between the proteinand the EOC can be isolated as such.

In the EPICs according to the present invention, the protein can beencapsulated entirely or partially by the EOC. It is preferred toencapsulate the protein entirely, i.e. the cavity is of a sizesufficiently large to accept the entire protein therein.

The EPICs according to the present invention can, in principle,accommodate any protein which has a physiological or pharmacologicalactivity. These are known to the person skilled in the art. Importantproteins can be found in standard text books which are known to theskilled artisan.

Relevant therapeutic proteins and polypeptides which can be encapsulatedaccording to the present invention are: ACTH, adenosine deaminase,agalsidase, albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinaseinhibitor (API), alteplase, anistreplase, ancrod serine protease,antibodies (monoclonal or polyclonal, and fragments or fusions),antithrombin III, antitrypsins, aprotinin, asparaginases, biphalin,bone-morphogenic proteins, calcitonin (salmon), collagenase, DNase,endorphins, enfuvirtide, enkephalins, erythropoietins, factor VIIa,factor VIII, factor VIIIa, factor IX, fibrinolysin, fusion proteins,follicle-stimulating hormones, granulocyte colony stimulating factor(G-CSF), galactosidase, glucagon, glucocerebrosidase, granulocytemacrophage colony stimulating factor (GM-CSF), gonadotropin chorionic(hCG), hemoglobins, hepatitis B vaccines, hirudin, hyaluronidases,idurnonidase, immune globulins, influenza vaccines, interleukins (1alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-1 receptor antagonist(rhIL-1ra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c, beta 1a,beta 1b, gamma 1a, gamma 1b), keratinocyte growth factor (KGF), lactase,leuprolide, levothyroxine, luteinizing hormone, lyme vaccine,natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF,pepsin, platelet activating factor acetylhydrolase (PAF-AH), prolactin,protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD),somatropins (growth hormone), somatostatin, streptokinase, sucrase,tetanus toxin fragment, tilactase, thrombins, thymosin, thyroidstimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNFreceptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urate oxidase,urokinase, vaccines.

Preferred proteins are antibodies, calcitonin, G-CSF, GM-CSF,erythropoietins, hemoglobins, interleukins, insulins, interferons, SOD,somatropin, TNF, TNF-receptor-IgG Fc.

The most preferred proteins are erythropoietins, interferons, insulins,somatropins and hemoglobins.

It is understood that the invention is not restricted to therapeuticproteins. Protection from aggressive environments is also desirable forother proteins such as amylases, proteases, peptidases, xylanases,lipases, lipoxygenases, cellulases, pectinases, phytases,oxidoreductases applied in industrial processes such as food and animalfeed applications, as cleaning compounds in laundry detergents,dishwashing detergents, in the manufacture of chemicals such as alcohol,steroids and antibiotics, amino acids, proteins, trigylcerides,phospholipids, and for textile, leather and fur applications, especiallyin the prebleaching of pulp.

All proteins, in particular those cited beforehand, can be encapsulatedin a macrocyclic structure according to the present invention, to resultin an MPIC, or in a dendrimer resulting in a DPIC.

The size of the cavity in the EOCs (i.e. the proteophors, macrocyclicstructures and dendrimers) according to the present invention needs tobe adapted to the proteins diameter. The size should be larger than thediameter of the smallest sphere that can be drawn around a correctlyfolded protein. From this diameter estimation, the length of thecorresponding molecular chain in the EOC host can be calculated. Inorder to encapsulate insulin (approximately 3 nm diameter), a chain ofat least 5 nm length that can fold into a halfcyclic conformation needsto be present in the EOC.

In a preferred embodiment of the present invention, the EMCs accordingto the formula (II) contain capping groups (C) which are arranged suchthat a dendritic structure of the EOC results. When such an EMC enclosesa protein, dendrimer-protein inclusion compounds DPIC according to theformula (V) wherein a dendrimer (VI) encapsulates the protein result.Such DPICs are a part of the present invention. In the followingformulae (V) and (VI) C denotes a capping group as defined beforehandand the other symbols have the meanings defined for formula (I).

Embodiments of the capped EOCs having three and four EMCs are shown inthe formulae (VII) and (VIII) below.

The DPICs according to the general formulae (V) to (VIII) show a 1:1ratio protein/dendrimer. The DPICs are soluble in water. In general, thecavity of the dendrimer also comprises water, in addition to theprotein.

Dendrimers are known to the person skilled in the art. Reference is madeto: Dendrimer II Architecture, Nanostructure and SupramolecularChemistry, Springer Verlag 2000, F. Vögtle Editor. Dendrimers are basedon repeating hyperbranched structures emanating from a central core(U.S. Pat. No. 4,507,466). These synthetic macromolecules are assembledin a stepwise fashion, with each reaction cycle adding another layer ofbranches (dubbed “generation”). Dendrimers are synthetically accessed bystepwise, divergent “bottom-up” or convergent “top-down” synthesis.Central structural component is the core unit from which hyperbrancheddendrimers extend in a radially symmetric fashion.

The dendrimers according to the present invention may contain, in thecapping groups, centers branching into two, three, four, or moredirections, preferably two.

The length of dendritic chains may be identical or vary between chainsof one dendrimer. Preferred chain lengths for individual dendrimers areup to 5000 bonds.

By the choice of appropriate capping groups, it is perfectly possible toprotect the encapsulated protein form the attack of e.g. antibodies orthe elimination by the kidney or the liver.

The capping groups C have been defined above, which definition alsoapplies here.

In all formulae (V) to (VIII) shown beforehand, an EMC can carry onecapping group C (as shown in the formulae) or more than one, i.e. 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or even more capping groups C.

In a preferred embodiment of the present invention, some capping groupsin dendrimers according to the invention contain branchedheterofunctional units carrying at least one thio-succinimido moiety.Thio-succinimido groups are the result of a reaction between a maleimidoand a thiol group and can be obtained under mild conditions, provinguseful for the synthesis of hyperbranched or dendritic structures.Further units which can be used in the synthesis of the dendrimersaccording to the present invention comprise tris-(2-maleimidoethyl)amineand hydroxysuccinimide ester (EP-A 0 618 192).

Most useful for the divergent assembly of hyperbranches or dendrimersare heterofunctional reagents carrying both a maleimido as well as aprotected thiol group. Such reagents may be employed for repeatedstepwise synthesis in a similar fashion as for instance protected aminoacids or protected nucleosides.

It was found that the dendrimers according to the present invention canefficiently be formed of multidentate compounds containing only onemaleimide group and a number of protected thiols, in a divergentsynthesis approach. The monomaleimido-tetrathio-dendrimer compoundsaccording to the present invention are of the general formulaM-A-(S-Pg)_(n), with the following meanings: M: maleimido, A: spacer, S:sulfur, Pg: thiol protecting group, n: 2 to 200.

Suitable thiol protecting groups: benzyl, 4-methoxybenzyl,2,4-dimethoxybenzyl-, 2,4,6-trimethoxybenzyl-(Tmob),4,4′-dimethoxyphenylmethyl-(diMpm), trityl-, 4-methoxytrityl-(Mmt),4,4′-dimethoxytrithyl-(DMTr), 4,4′,4″-trimethoxytrityl-(TMTr),tert.-butyl-MeCONHCH2-(Acm), PhCH2CONHCH2-(PhAcm), MeOCOS-(Scm),BzlOCOS-(SZ) PhN(Ne)COS-(Snm), TrtS-, 2-pyridinesulfenyl-,2-(3-nitropyridinesulfenyl),4,5,6-trimethoxy-2,3-dihydro-7-benzofuranylmethyl-(Tmbf),2-(2,4-dinitrophenyl)ethyl-(Dnpe), 9H-xanthen-9-yl-Xan),2-methoxy-9H-xanthen-9-yl-(Moxan), Fmoc-S-sulfonate.

Multidentate compounds are water soluble and the conjugation reactionswill not compromise the biomolecule's structural integrity orbioactivity.

According to the present invention, the core of the DPIC is formed bythe protein to be encapsulated which is connected (conjugated) to thepolymer backbone by a suitable linker, in general one of the linkerslisted above.

After the cleavage of the linker, a DPIC results in which the protein isnot connected to the dendrimer via a linker (a chemical bond), but theprotein is held within the cavity defined by the dendrimer. Theresulting DPICs are an object of the present invention. It depends onthe release kinetics of the protein if the DPIC having no bond betweenthe protein and the dendrimer can be isolated as such.

In a further embodiment of the present invention, the EOCs comprise asecond branching group B identical or different from the first branchinggroup B to which the EMCs are connected, resulting in a cavity which ishorizontally locked and vertically open. Thus, the encapsulation isrealized by a macrocycle-protein inclusion compound MPIC according tothe formula (IX)

containing a protein P and a macrocyclic structure encapsulating theprotein totally or partially according to the general formula (X)

wherein the symbols in formulae (IX) and (X) have the followingmeanings:

-   -   B: branching unit (basic unit, core) containing at least one        branching center Bc and at least two branching functional groups        Bfg connected to or capable of reacting with an encapsulating        unit EMC;    -   EMC: encapsulating molecular chain;    -   L: linker containing at least one functional group Lfg which is        connected to the protein P or capable of connecting with        functional groups present on the protein P under the formation        of a chemical bond;    -   l: 1, 2, 3, 4, 5, 6, 7, 8 or 9, preferably 1, 2, 3, 4 or 5, in        particular 1, 2 or 3;    -   P: pharmacologically active protein.

The MPICs according to the general formula (IX) show a 1:1 ratioprotein/macrocyclic structure. The MPICs are soluble in water. Ingeneral, the cavity of the macrocyclic structure also comprises water,in addition to the protein.

The EMCs contain hydrophilic groups, in an appropriate ratio and amountwith respect to hydrophobic groups which may be present in themacrocyclic structure, to render the latter water soluble.

B, EMC, L and P are in accordance with the definitions given above.

The macrocyclic structure and the MPIC according to the presentinvention comprise at least two EMCs (l=1). The macrocyclic structurecan however comprise 3 (l=2), 4 (l=3), 5 (l=4), 6 (l=5) or even up to 10EMCs. Some embodiments are depicted in the formulae (XI) and (XII)below.

The EMCs can all be identical, partly identical (partly different) orentirely different from each other.

The EMCs can carry capping groups C, as defined above, on theirbackbone. Appropiate capping groups are sterically demanding groups. Themacrocyclic structures of the present invention are not rigid, and theEMCs, due to the rotation around the bonds connecting them to thebranching units B, may swing to one side, resulting in a staggering andcrowding on one side of the macrocyclic structure, and opening of one ormore sides of the cavity. Capping groups prevent sterical proximity ofthe EMCs. If the EMCs come too close to one another, insufficientprotection of the encapsulated protein from the attack of enzymes,antibodies or the like may result. Furthermore, the protein may leavethe cavity (if the linker is broken) through the gap, resulting in anundesired release kinetics of the protein. In case capping groups arepresent, the following structures (XIII) and (XIV) result.

The figures (XV) and (XVI) below show macrocyclic structures havingcapping groups on the three and four EMCs of the respective macrocyclicstructure.

In all formulae (XI) to (XVI) shown beforehand, an EMC can carry onecapping group C (as shown in the formulae) or more than one, i.e. 2, 3,4, 5 or even more capping groups C.

Examples for EPICs, DPICs and MPICs wherein the linker has been cleavedand the protein is not held within the cavity by covalent bonds aredepicted in the formulae (XVII) to (XX) below, which include examples inwhich traces of the linker remain at the BOC, dendrimer or macrocyclicstructure, and examples wherein the linker has totally been removed.

Modified proteins containing a linker are a part of the presentinvention. Within this embodiment, it is preferred if the linker is aprodrug linker or a traceless linker, more preferably traceless prodruglinker.

After the cleavage of the linker, a MPIC results in which the protein isnot connected to the macrocyclic structure via a linker (a chemicalbond), but the protein is held within the cavity defined by themacrocyclic structure. The resulting MPICs are an object of the presentinvention. It depends on the release kinetics of the protein if the MPIChaving no bond between the protein and the macrocyclic structure can beisolated as such.

The EPICs, MPICs and the DPICs of the present invention are synthesizedfrom the protein and the EOC, macrocyclic structure and dendrimer,respectively, by a combination of solid-phase and solution synthesismethods known to the person skilled in the art.

The host molecule may be equipped with the linker moiety and be attachedto the protein in one single reaction step (convergent synthesis).Alternatively, the linker-protein conjugate may be reacted with thebranching unit B contained in the EOC, dendrimer or macrocyclic backbonestructure. In another, divergent manifestation of the process,protein-linker-branching unit conjugate is reacted with presynthesizedEOCs, macrocyclic structures or dendrimers. In an even more divergentapproach, the dendrimers, macrocyclic structures or EOCs are assembledin a stepwise fashion in an extension of the centralprotein-linker-branching unit structure.

Efficient encapsulation is demonstrated by antibody binding studies.Antibodies against therapeutic proteins are high-affinity,high-selectivity probes. Steric shielding of the protein prevents accessof the antibodies to the epitopes for molecular recognition. Antibodybinding may be conveniently and reliably measured by methods known tothe person skilled in the art, preferably immunoprecipitation or, asexemplified here, by label-free surface plasmon resonance scanning. In astudy involving various insulin derivatives including insulinsconjugated to different PEG reagents, and three monoclonalanti-insulins, complete prevention of antibody recognition was onlyachieved if insulin was complexed with a macrocyclic structure accordingto the present invention. In order to eliminate any bias by insulinrelease, non-cleavable covalent PEG or EOC conjugates were employedrespectively.

The resulting EOC self-organizes into a biomolecule-containing void byconformationally folding around the protein. This arrangement may bedriven by sterical constraints or by chemical reactions or both.

Eventually, the linker is cleaved, and a host-guest complex is obtained.Linker cleavage may be performed in vitro to generate a non-covalentcomplex. Alternatively, linker cleavage may occur in a prodrug approachin vivo after administration. Dissociation kinetics of the complex maybe governed by linker hydrolysis or protein effusion through themolecular matrix of the EOC, dendrimer, or macrocyclic structure, or acombination of both.

In the presence of the protein, the complex is characterized by awell-defined cavity-forming chemical structure and precisestoichiometry. After protein release, the host molecule may adoptvarious conformations due to its structural flexibility, for whichreason protein release is essentially irreversible.

The formulae (XXI) to (XXIV) below show DPICs and MPICs wherein the bondbetween the linker and the protein has been cleaved; in two cases,traces of the linker remain at the EOC, dendrimer or macrocyclicstructure, and in two cases the linker has totally been removed.

The present invention also relates to method for selectively deliveringa protein to a target, which method comprises

-   -   providing an encapsulated protein;    -   bringing the encapsulated protein into contact with a body        liquid containing the target.

The encapsulated protein can be formulated into a drug, optionallytogether with one or more pharmaceutically acceptable carriers. The drugcan contain one or more encapsulated protein types.

Drug containing at least one encapsulated protein according to thepresent invention and optionally one or more pharmaceutically acceptablecarriers are also an object of the present invention

The present invention will now be illustrated by the following,non-limiting examples.

DESCRIPTION OF THE DRAWINGS

FIG. 1: Size exclusion chromatograms of a) native Hb, b) 39, c) reactionmixture 40, and d) purified covalent Hemoglobin MPIC 40. UV signals wererecorded at 280 nm.

FIG. 2 Size exclusion chromatograms of a) 41, b) 42 and c) Hb releasedfrom 42. UV signals were recorded at 280 nm.

FIG. 3: Size exclusion chromatograms of a) reaction mixture resultingfrom linker cleavage procedure performed on 50a, b) Insulin MPIC 50a. UVsignals were recorded at 280 nm.

FIG. 4: In vitro release of insulin from Insulin MPIC 64 or 65,respectively. Kinetics were determined by HPLC using UV detection at 215nm.

FIG. 5: Release of insulin from dendrimer prodrugs 90 (top) and 91(below). Free insulin was quantified by HPLC using UV detection at 215nm.

FIG. 6: Binding of insulin or insulin conjugates to immobilized murineanti insulin antibodies (clones 8E2, C7C9 and 7F8).

EXAMPLES

Materials and Methods

Materials

Fmoc-amino acids, resins and TBTU were purchased from Novabiochem andare named according to the catalogue. Fmoc-Ado-OH was obtained fromNeosystem (France) and Fmoc-PP—OH from Polypure (Norway). All additionalchemicals were purchased from Sigma Aldrich. Human insulin was from ICNBiomedicals (USA). Maleimide-PEG5k, Maleimide-PEG20k andMaleimide-PEG2x2Ok were obtained from Nektar.

Reaction Medium

Solid phase synthesis was performed on TentaGel TGR or Sieber amideresin with a loading of 0.2 mmol/g or 0.5 mmol/g, respectively. Syringesequipped with polypropylene frits were used as reaction vessels.

Standard Coupling Cycle for Fmoc-Protected Amino Acids

For Fmoc protecting-group removal, resin was repeatedly (three times, 4min each) agitated with 2/2/96 (v/v/v) piperidine/DBU/DMF and repeatedly(six times) washed with DMF. Coupling of Fmoc-protected amino acids toresin was achieved by agitating the resin with 3 equivalents (eq) ofFmoc-amino acid, 3 eq TBTU and 6 eq DIEA in DMF for 60 min. Finally, theresin was repeatedly (five times) washed with DMF.

Standard Cleavage Protocol for TentaGel TGR Resin

Upon completed synthesis, resin was washed with DCM, dried in vacuo andtreated with 95/5 (v/v) TFA/TES. After evaporation, compounds werepurified by preparative RP-HPLC (Waters 600).

Analysis

Mass spectrometry (MS) was performed on a waters ZQ 4000 ESI instrumentand spectra were, if necessary, interpreted by waters software MaxEnt.

Size exclusion chromatography were performed using a Waters 600 systemsequipped with either a Superdex 75 or Superdex 200 column (Pharmacia) ormanually using a PD10 column (Pharmacia).I—Synthesis of Bifunctional Linkers

0.7 g TentaGel TGR resin was soaked in THF and incubated for 60 min witha solution of 4 ml of 0.5 M chloroformic acid-4-nitrophenyl ester and0.5 M DIEA in THF, washed with THF and dried. 4 ml of a suspension of0.3 M cystamine-dihydrochloride and 0.7 M DIEA in DMSO were added to theresin and agitated for 90 min. Resin was washed with DMSO and DMF, and 3eq maleimidopropionic aid and 3 eq DIC in DMF were added and agitatedfor 60 min

After washing the resin with DMF and DCM, compound 1 was cleaved fromthe resin and purified by RP-HPLC.

MS (MW calculated) 1: 346 g/mol (346.4 g/mol)

Cystamine-dihydrochloride was suspended in 1/1 (v/v) DMSO/DMF and mixedwith 2 eq maleimidopropionic acid, 2 eq DIC and 2 eq DIEA. Thesuspension was agitated for 2 h at room temperature (RT) and afteracidification with formic acid, compound 2 was purified by RP-HPLC.

MS (MW calculated) 2: 454 g/mol (454.5 g/mol)

Cysteamine hydrochloride was dissolved in TFA and 0.5 eq Mmt-chloridewere added. After 30 min, TFA was removed under nitrogen flow and theresidue was taken up in pyridine. After adding a solution of 0.2 MNa₂CO₃, product was extracted with ether and dried over Na₂SO₄.Following filtration, solvents were removed using a rotary evaporatorand compound 3 was obtained as a highly viscous oil.

3 was reacted with 4-hydroxymethyl-phenoxyacetic acid (HMPA) and 1 eqHOBT/DIC for 30 min. After purification by RP-HPLC, compound 4 wasneutralized by DIEA and lyophilized.

4 was reacted with 5 eq chloroformic acid-4-nitrophenyl ester and 10 eqDIEA in dioxane for 2 h. Subsequent purification by RP-HPLC gave product5.

MS [MNa]⁺ (MW+Na calculated) 5: 700 g/mol (701 g/mol)

300 mg of 4-hydroxy-3-methoxy-phenylacetic acid and 570 μl DIEA weredissolved in 5 ml DCM and added to 0.5 g 2-chlorotrityl-chloride resin(1.5 mmol/g). The suspension was agitated for 1 h at RT and resin waswashed with DCM. Resin was resuspended in a solution of 190 mg3-maleimidopropionic acid, 333 mg MSNT and 73 μl N-methyl imidazole in 3ml DCM and agitated for 1 h. After washing of the resin with DCM,cleavage was performed by agitation of the resin for 30 min in 10 ml 4/1(v/v) DCM/TFA. Solvent was removed under nitrogen flow and compound 6was purified by RP-HPLC.

MS [MNa]⁺ (MW+Na calculated): 356 g/mol (356 g/mol)

512 mg Fmoc-3-aminopropionic acid and 570 Ill DIEA were dissolved in 5ml DCM and added to 0.5 g 2-chlorotrityl-chloride resin (1.5 mmol/g).The suspension was agitated for 1 h at RT and resin was washed with DCM.After Fmoc-removal with 96/2/2 (v/v/v) DMF/DBU/piperidine and washing ofthe resin with THF, 4 ml of a solution of 0.5 M chloroformicacid-4-nitrophenyl ester and 0.5 M DIEA in I1F were incubated with theresin for 30 min. Resin was washed with THF and DMF and agitated for 30min with a solution of 1 M cystamine in DMF and washed with DMF.Subsequently, resin was agitated for 15 min in a solution of 2/1/1(v/v/v) DMF/acetic anhydride/pyridine. After washing the resin with DMFand DCM, compound 7 was cleaved by agitation for 30 min in 10 ml of 4/1(v/v) DCM/TFA. After evaporation of solvent under nitrogen flow, product7 was purified by RP-HPLC.

MS (MW calculated): 309 g/mol (309 g/mol)

Mmt-chloride (1 eq) and mercaptopropionic acid (1.1 eq) were taken up inTFA and incubated for 30 min. TFA was removed under nitrogen flow. Theproduct was dissolved in pyridine, diluted in water, acidified by aceticacid and extracted in ether. The ether phase was separated and driedover Na₂SO₄. Solvent was removed and product 8 was purified by RP-HPLC.

Compound 8 and DMAP (2.5 eq) were dissolved in dry DCM and 2 eq of EDCHCl were added at 0° C. The solution was stirred for 14 hat RT andwashed with sodium acetate buffer (0.25 M, pH 4.5). The organic phasewas dried over Na₂SO₄, concentrated and compound 9 was purified bysilica gel column chromatography using heptane/acetic acid ester (1/1)as mobile phase.

MS (MW calculated) 9: 479 g/mol (479.7 g/mol)

Compound 9, 4-hydroxy-3-methoxy benzylalcohol (7 eq) and DMAP (7 eq)were refluxed in DCM for 2 h under nitrogen atmosphere. Afterneutralization with acetic acid, the solution was concentrated andcompound 10 was purified by RP-HPLC:

MS [M+Na]⁺ (MW+Na calculated) 10: 537 g/mol (537,6 g/mol)

Compound 10, chloroformic acid-4-nitrophenyl ester (10 eq), and DIEA (20eq) were stirred in dry dioxane for 3 h at 40° C. under nitrogenatmosphere. After addition of acetic acid (25 eq) the mixture wasconcentrated and compound 11 purified by RP-HPLC:

MS [M+Na]⁺ (MW+Na calculated) 11: 702 g/mol (702.7 g/mol)II—Synthesis of Bifunctional Macrocyclic Structures

Employing the standard protocol for solid-phase synthesis, the aminoacids Fmoc-Cys(S-tBu)-OH, Fmoc-Lys(Fmoc)-OH, two units of Fmoc-PP—OH andFmoc-Cys(Trt)-OH were coupled to TGR resin. After final Fmoc-removal,resin was treated with 2/1/1 (v/v/v) DMF/acetic anhydride/pyridine for15 min. Compound 12 was cleaved from the resin and purified by RP-HPLC.

MS (MW calculated) 12: 3024.8 g/mol (3025.8 g/mol)

Employing the standard protocol for solid-phase synthesis, the aminoacids Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Fmoc) and two units of Fmoc-PP—OH werecoupled to TGR resin. After final Fmoc removal, the resin was treatedfor 30 min with 5 eq maleimidopropionic acid and 5 eq DIC in DMF.Removal of the Mtt-protecting group was achieved by repeated washing ofthe resin with 99/1 (v/v) DCM/TFA until the solution remained colorless.Resin was washed with DCM, 1% DIEA in DMF (briefly) and DMF.Subsequently, resin was incubated for 25 mmn with a solution of 0.5 Mbromoacetic acid and 0.5 M DIC in DMF.

After cleavage, compound 13 was purified by RP-HPLC.

MS (MW calculated) 13: 3095.2 g/mol (3095.5 g/mol)

12 and 13 were mixed at an equimolar ratio and concentration wasadjusted to 35 μM by addition of 0.1% aqueous TFA. The pH of thesolution was adjusted to 7.5 with 0.1 M phosphate buffer (pH 7.5). After20 min the pH of the mixture was brought to 2.0 by addition of formicacid, and product 14 was purified by RP-HPLC and lyophilized.

MS (MW calculated) 14: 6120 g/mol (6121.1 g/mol).

2 eq 14 were mixed with 1 eq Ac-Cys-Lys-Cys-NH₂ (obtained according tothe standard protocol for solid phase synthesis) at a concentration of350 μM and the pH of the solution was adjusted to 8.0 with phosphatebuffer. After 90 min the reaction was quenched by acidification withacetic acid, and product 15 was purified by RP-HPLC.

MS (MW calculated) 15: 12472 g/mol (12474.2 g/mol)

S-tBu protecting groups in 15 were removed by reduction with 100 mM DTTin phosphate buffer (H 8.0). After 3 h the pH of the solution wasadjusted to 2.0, and compound 16 was purified by RP-HPLC.

MS (MW calculated) 16: 12295 g/mol (12297.8 g/mol)

16 was mixed with 1 eq Ac-Dpr(Mal)-Lys-Dpr(Mal)-NH₂ (obtained accordingto the standard protocol for solid phase synthesis usingFmoc-Dpr(ivDde)-OH) and concentrations were adjusted by addition ofwater to 15 μM. Subsequently, pH was adjusted to 7.5 with 0.1 Mphosphate buffer (pH 7.5) and stirred for 20 min. The pH of the solutionwas brought to 2.0 by formic acid, and compound 17 was purified byRP-HPLC and lyophilized.

MS (W calculated) 17: 12955 g/mol (12959.5 g/mol)

17 was dissolved in DMF and agitated for 30 min with a solution of 20 eqmaleimidopropionic acid, 30 eq DIEA and 20 eq DIC in DMF. Subsequently,the solution was acidified with formic acid, diluted with water and 18was purified by RP-HPLC and lyophilized.

MS (MW calculated) 18: 13257 g/mol (13261.7 g/mol)

17 was adjusted to a concentration of 300 μM in 0.1 M phosphate buffer(pH 7.0) and 15 eq SPDP in DMSO were added. The resulting suspension wasagitated for 20 min at RT. 10 mM TCEP were added, and the cocktail wasagitated for another 20 min at RT. Product 19 was purified by RP-HPLC.

MS (MW calculated) 19: 13132 g/mol (13135,7 g/mol)

Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Fmoc)-OH and Fmoc-PP—OH were coupledaccording to the standard protocol for solid phase synthesis. After Fmocremoval, resin was incubated with 6 eq male imidopropionic acid and 6 eqDIC for 30 min. Following cleavage from resin, compound 20 was purifiedby RP-HPLC.

MS (MW calculated) 20: 1774 g/mol (1775 g/mol)

Compounds 21a and 21b were obtained by solid phase synthesis accordingto protocol II-1) for product 12.

MS (MW calculated) 21a: 1825 g/mol (1826 g/mol)

MS (MW calculated) 21b: 3024 g/mol (3026 g/mol)

Reactions of educts 20 and 21a to product 22a, and educts 20 and 21b toproduct 22b were performed according to protocol II-1) in analogy to thereaction of compounds 12 and 13 to product 14.

MS (MW calculated) 22a: 3600 g/mol (3601 g/mol)

MS (MW calculated) 22b: 4800 g/mol (4801 g/mol)

Compounds 22a or 22b, respectively, were dissolved in DMF and pH wasadjusted to 8.0 with DIEA. 6 eq Maleimidopropionic acid and 6 eq DICwere added and mixtures were incubated for 30 min to yield compounds 23aand 23b, respectively. Purification was performed by RP-HPLC.

MS (MW calculated) 23a: 3752 g/mol (3753 g/mol)

MS (MW calculated) 23b: 4950 g/mol (4952 g/mol)

2 eq of compound 23a or 23b respectively were mixed with 1 eqAc-Cys-Lys-Cys-amide (obtained according to the standard protocol forsolid phase synthesis). After adjusting the pH to 8.0 by addition of 0.5M phosphate buffer (pH 8.0) the solution was agitated for 10 min. Thereaction was quenched by addition of 10 eq DTT. After lyophilization theresidue was taken up in 1:1 (v/v) acetonitrile/50 mM phosphate buffer(pH 8.0). Removal of the S-tBu protecting group by reduction with 50 mMDTT for 2 h yielded products 24a and 24b, respectively. Purification wasby RP-HPLC;

MS (MW calculated) 24a: 7719 g/mol (7722 g/mol)

MS (MW calculated) 24b: 10120 g/mol (10121 g/mol)

Compounds 24a or 24b, respectively, were subjected to two additionalreaction steps to yield 25a or 25b, respectively, according to protocolII-1 and in analogy to the reaction of compounds 16 and 17 to product18.

MS (MW calculated) 25a: 8686 g/mol (8686 g/mol)

MS (MW calculated) 25b: 11085 g/mol (11085 g/mol)

Compound 26 was obtained according to the standard protocol for solidphase synthesis. The amino acids Fmoc-Cys(StBu)-OH, Fmoc-Lys(Fmoc)-OH,Fmoc-PP—OH, Fmoc-Lys(Boc)-OH, Fmoc-PP—OH and Fmoc-Cys(Mmt) were coupledto Sieber amide resin. After final Fmoc-removal, resin was treated witha solution of 2/1/1 (v/v/v) DMF/acetic acid anhydride/pyridine for 15min, washed with dichloromethane and dried in vacuo. Cleavage wasperformed by repeated treatment (15 times) of the resin for 2 min with asolution of 97/1/2 (v/v/v) dichloromethane/TFA/TES. Collectedsupernatant was mixed and buffered with 1 eq pyridine (vs. TFA). Afterconcentrating the mixture, compound 26 was purified by RP-HPLC.

MS (MW calculated) 26: 3482 g/mol (3482 g/mol)

Compound 27 was obtained according to the standard protocol for solidphase synthesis. Starting from Sieber amide resin, the amino acidsequence Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Fmoc)-OH, Fmoc-PP—OH,Fmoc-Lys(Boc)-OH, Fmoc-PP—OH and Fmoc-Lys(Boc)-OH was assembled. Afterfinal Fmoc-removal, the resin was reacted for 30 min with 6 eqmaleimidopropionic acid and 6 eq DIC, washed with dichloromethane anddried in vacuo. Cleavage was performed by repeated (15 times) treatmentof the resin for 2 min with a solution of 97/1/2 (v/v/v)dichloromethane/TFA/TES. Collected supernatant was mixed and bufferedwith 1 eq pyridine versus TFA. After concentrating the mixture, compound27 was purified by RP-HPLC.

MS (W calculated) 27: 3888 g/mol (3887 g/mol)

Compound 28 was obtained from educts 26 and 27 in analogy to thesynthesis of 24 from educts 20 and 21 according to protocol II-3.

Cyclization of 28 to 29 was performed in analogy to cyclization ofcompound 16 according to protocol II-1.

For analysis, a sample was treated with TFA to effect removal of theBoc-protecting groups.

MS (MW calculated without Boc-groups) 29: 14717 g/mol (14720 g/mol)

29 were reacted with 10 eq 6 and 10 eq DIC in DMF for 30min. Product waspurified by RP-HPLC and lyophilized. Boc-protecting groups were removedby incubation for 30 min in 1/1 (v/v) TFA/DCM. Solvent was evaporatedunder nitrogen flow. Extraction of the residue with water and subsequentlyophilization yielded 30.

MS (MW calculated): 15352 g/mol (15360 g/mol)

Product 31 was obtained by reacting 30 with 30 eq 7 and 30 eq DIC in DMFfor 45 min. Product was purified by RP-HPLC and lyophilized.

MS MW calculated): 18851 g/mol (18847 g/mol)

Compound 32 was obtained according to the standard protocol for solidphase synthesis. The amino acids Fmoc-Cys(StBu)-OH, Fmoc-Lys(Fmoc)-OH,Fmoc-PP—OH and Fmoc-Cys(Mmt)-OH were coupled to Sieber amide resin.After final Fmoc-removal, the resin was incubated with a solution of2/1/1 (v/v/v) DMF/acetic acid anhydride/pyridine for 15 min, washed withdichlorornethane, and dried in vacuo. Cleavage from the resin wasafforded by treatment with 97/1/2 (v/v/v) Dichlormethan/TFA/TES for 30min. After concentration, product 32 was purified by RP-HPLC

Compound 33 was obtained according to the standard protocol for solidphase synthesis. The amino acids Fmoc-Lys(Mtt)-OH, Fmoc-Lys(Fmoc)-OH,Fmoc-PP—OH and Fmoc-Cys(Trt)-OH were coupled with Sieber amide resin.After final Fmoc-removal, the resin was incubated with 6 eqmaleimidopropionic acid and 6 eq DIC for 30 min, washed withdichloromethane, and dried in vacuo. Cleavage from the resin wasafforded by treatment with 99/1/(v/v) dichloromethane/TFA for 15 min andrepeated washing with DCM. Pooled eluates were buffered with 0.75 eqpyridine (versus TFA) and solvent was removed in vacuo. Product waspurified by RP-HPLC.

MS (MW calculated) 33: 2466 g/mol (2466 g/mol)

Synthesis of 34 was performed in analogy to the cyclization of compound25 according to protocol II-3, except for the use of Ac-Lys(Mal)-Lys-Lys(Mal)-NH₂ instead of Ac-Dpr(Mal)-Lys-Dpr(Mal)-NH₂. Foranalysis, a sample of 34 was treated with 48/50/2 (v/v/v) TFA/DCM/TESfor 10 min to effect removal of the Trt-protecting groups.

MS (MW calculated without Trt groups) 34: 9183 g/mol (9183 g/mol)III—Synthesis of Proteophore-Capping Reagents

Compound 35 was obtained according to the standard protocol for solidphase synthesis. The amino acids Fmoc-Lys(Boc)-OH, Fmoc-Lys(Fmoc)-OH andFmoc-Lys(Fmoc)-OH were coupled to TGR resin, and Mmt-3-mercaptopropionic(8) acid was used as the terminal building block. After resin cleavage,product was purified by RP-HPLC.

MS (MW calculated) 35: 881.5 g/mol (882 g/mol)

Compound 35 and 4.1 eq Maleimide-PEG5k were dissolved in 0.1 M sodiumphosphate buffer (pH 7.0) and stirred for 30 min. Excess Maleimide-PEG5kwas reacted with mercaptoethanol and product 36 was purified by RP-HPLCand lyophilized.

Compound 36 was taken up in DMF, 6 eq maleimidopropionic acid and 6 eqDIC in DMF were added and the mixture was agitated for 30 min. Theproduct was purified by RP-HPLC and characterized by size exclusionchromatography (Superdex 200 column, flow rate: 0.75 ml/min)

SEC (retention time) 36: 16 min

Compound 38 was obtained according to the standard protocol for solidphase synthesis. The amino acids Fmoc-Dpr(ivDde)-OH, Fmoc-Lys(Fmoc)-OH,Fmoc-Lys(Fmoc)-OH and two units of Fmoc-PP—OH were coupled to TGR resin.After final Fmoc-removal, resin was incubated with a solution of 2/1/1(v/v/v) DMF/acetic acid anhydride/pyridine for 15 min. Removal of theivDde-protecting group was afforded by repeatedly agitating (threetimes) the resin for 5 min with 98/2 (v/v) DMF/hydrazine. The resin waswashed with DMF and treated for 30 min with a solution of 6 eqmaleimidopropionic acid and 6 eq DIC in DMF.

After cleavage from the resin the product was purified by RP-HPLC.

MS (MW calculated) 38: 5600 g/mol (5603 g/mol)

IV-1) Modification of Hb Cys93(β) with Linker 1

Human hemoglobin (Hb) was adjusted to a concentration of 10 mg/mi in 0.1M sodium phosphate buffer (H 7.5), 5 eq 1 were added and the mixture wasagitated for 30 mi at RT. Excess 1 was removed by size exclusionchromatography PD10 column).

MS alpha-subunit, unmodified (MW calculated): 15121 g/mol (15127 g/mol)

MS beta-subunit (MW calculated): 16208 g/mol (16214 g/mol)

Cleavage of the disulfide bond of the conjugated linker was effected byreduction of the modified Hb for 30 min in 5 mM TCEP (pH 7.5). Product39 was purified by SEC (Superdex 200).

MS alpha-subunit, unmodified (MW calculated) 39: 15121 g/mol (15127g/mol)

MS beta subunit (MW calculated) 39: 16090 g/mol (16096 g/mol)

IV-2) Conjugation to Bis-Maleimido-Macrocycic Structure 18

Compound 39 was adjusted to a concentration of 20 μM in 0.1 M phosphatebuffer (pH 7.5) and 2 eq of 18 were added. After incubation for 20 minat RT, the resulting Hb MPIC 40 was purified by SEC (Superdex 200column). FIG. 1 displays size exclusion chromatograms of native Hb, 39,the reaction mixture of 39 and Hb, and purified product 40.

MS alpha subunit, unmodified (MW calculated) 40: 15122 g/mol (15127g/mol)

MS crosslinked beta subunits (MW calculated) 40: 45432 g/mol (45454g/mol)

V-1) Modification of Hb Cys93(β) with Linker 2

Hb was adjusted to a concentration of 10 mg/ml in 0.1 M sodium phosphatebuffer (pH 7.5). After addition of 10 eq 2 the solution was agitated for30 min at RT. Hb-conjugate 41 was purified by SEC (Superdex 200).

MS alpha subunit, unmodified (MW calculated) 41: 15123 g/mol (15127g/mol)

MS beta subunit (MW calculated) 41: 16318 g/mol (16322 g/mol)

V-2) Conjugation to Bis-Thiol-Macrocyclic Structure 19

The concentration of 41 was adjusted to 20 μM in 0.1 M phosphate buffer(pH 7.5). After addition of 2 eq 19 the solution was incubated for 45min at RT. The Hemoglobin MPIC 42 was purified by SEC (Superdex 200).

MS alpha subunit (MW calculated) 42: 15125 g/mol (15127 g/mol)

MS beta subunit (MW calculated) 42: 45776 g/mol (45780 g/mol)

In order to prove the reversibility of the conjugation, product 42 wastreated with 5 mM TCEP in 0.1 M phosphate buffer (pH 7.5). Thequantitative release of Hb from the MPIC was assessed by LC/MS.

MS beta subunit (MW calculated) 42: 16091 g/mol (16096 g/mol)

FIG. 2 displays size exclusion chromatograms of 41, 42 and of Hb (44)released from 42.

Structural element 37′ represents the succinimidyl-containing product ofthe Michael addition of the neighboring proteophore thiol to themaleimido group of 37.

VI-1) Conjugation of 39 to Bis-Maleimide-Macrocyclic Structure 31

The conjugation of 39 to 31 was performed according to protocol IV-2 inanalogy-to the preparation of 40. Disulfide-moieties associated with theproteophore were reduced by incubation in 10 mM TCEP (pH 7.5) for 30min. Product 45 was purified by SEC (Superdex 200 column).

VI-2) Modification of Hemoglobin MPIC with Mal-PEG4x5k (37)

Compound 45 was reacted with 30 eq 37 for 1 h at RT in 50 mM sodiumphosphate buffer (pH 7.0) and purified by SEC (Superdex 200, flow rate:0.75 ml/min).

SEC (retention time) 46: 11.2 minVII—Synthesis of an Insulin MPIC

Insulin was dissolved in DMSO and reacted with 1.1 eq (t-BOC)₂O for 60min to yield N^(αA1)-Boc-insulin 47. Purification was achieved byRP-HPLC. Enzymatic digestion of 47 with endo-GluC and subsequentcharacterisation of the resulting fragments by LCMS confirmedregioselective modification of the amino terminus of the A chain ofinsulin.

MS (MW calculated) 47: 5907 g/mol (5907 g/mol)

47 was dissolved in DMSO and incubated with 10 eq of activated linker 5for 5 h at pH 8-9. The pH was adjusted by addition of DIEA, ifnecessary. Subsequent RP-HPLC purification affordedN^(αA1)-Boc,N^(αB1),N^(εB29)-bis-(Mmt-thiollinker)-insulin 48. Afterlyophilization, the Mmt-protecting group was removed by incubation for30 min with 1/99 (v/v) TFA/DCM and productN^(αA1)-Boc,N^(αB1),N^(εB29)-bis-(thiollinker)-insulin 49 was purifiedby RP-HPLC.

MS (MW calculated) 49: 6450 g/mol (6442 g/mol)

A solution of 49 (20 μM) in 3/1 (v/v) 25 mM phosphate buffer (pH7.5)/acetonitrile was reacted with 1.1 eq of 25a or 25b, respectively,for 15 min to yield 50a or 50b, respectively. Purification was achievedby RP-HPLC.

MS (MW calculated) 50a: 15128 g/mol (15128 g/mol)

MS (MW calculated) 50b: 17527 g/mol (17527 g/mol)

Insulin was released from conjugates 50a or 50b, respectively, bycleavage of the linker moieties by incubation with 1:1 (v/v) DCM/TFA for15 min. These conditions also effected removal of the Boc-protectiongroup on the α-Aminogruppe of the insulin A-chain.

Educts 50a and 50b, linker cleavage mixtures containing released insulinand remaining macrocyclic structures 52a and 52b were characterized bySEC (Superdex 200, flow rate: 0.75 ml/min).

FIG. 3 displays size exclusion chromatograms of 50a and products of thelinker cleavage procedure. Three peaks were detected and identified byLCMS analysis. The peak at retention time 23.2 min contains insulin, thepeak at 21.6 min is EOC 52a. The peak at retention time 20.7 mincorresponds to EOC-insulin-monoconjugate.VIII—Synthesis of Insulin MPIC 57, 58 and 59

240 μl of a solution containing 0.64 M DIC, 0.56 M 8 and 0.29 MN-hydroxysuccinimide in DMF were preincubated for 30 min. 15 mg 47 weredissolved in 1 ml 1/1 (v/v) DMSO/water and mixed with 40 μl DIEA. Themixtures were combined and incubated for 1 h. Compound 53 was purifiedby RP-HPLC.

MS (MW calculated) 53: 6630 g/mol (6627 g/mol)

After lyophilization, Mmt- and Boc-protection groups were removed byincubation with 2/48/50 (v/v/v) TES/TFA/DCM for 30 min. Product 54 waspurified by RP-HPLC.

MS (MW calculated) 54: 5981 g/mol (5983 g/mol)

150 μl of a 3 mM solution of compound 54 in 1/1 (v/v) water/acetonitrilewere mixed with 270 μl of a 1.5 mM solution of macrocyclic structure 34in 1/1 (v/v) water/acetonitrile and diluted with 20/80 (v/v)acetonitrile/water to a total volume of 22 ml. Subsequently, pH wasadjusted to 7.5 by addition of 0.5 M phosphate buffer pH 8.0.

After 15 min incubation, compound 55 was purified by RP-HPLC andlyophilized.

Removal of Trt-protecting groups from compound 55 was effected byincubation for 30 min in 2/58/40 (v/v/v) TES/TFA/DCM. A two-steppurification procedure employing RP-HPLC and SEC (Superdex 75, flowrate: 0.75 ml/min) yielded product 56.

MS (MW calculated) 56: 15167 g/mol (15166 g/mol)

Structural element R represents the succinimidyl-containing product ofthe Michael addition of the neighboring proteophore thiol to themaleimido group of 37, 38 or N-ethyl maleimide, respectively. Aliquots(60 nmol) of compound 56 were reacted with 10 eq N-ethyl-maleimide, or37 or 38, respectively, for 15 min in 500 μl 1/4 (v/v) acetonitrile/i 00mM phosphate buffer (pH 8.0). Subsequent purification by SEC (Superdex200, flow rate: 0.75 ml/min) yielded products 57, 58 or 59,respectively. Compound Retention time 57 20.8 min 58 16.8 min 59 13.5minIX—Synthesis of Insulin MPIC 64 and 65

47 was mixed with 4 eq 11 in DMSO. The solution was adjusted to pH 8.0by addition of DIEA and stirred for 2.5 h at RT under nitrogenatmosphere. RP-HPLC-purification and lyophilization gave compound 60.

MS (MW calculated) 60: 6991 g/mol (6991 g/mol)

Removal of Trt-protection of 60 was afforded by stirring the compound inTFA/TES 95/5 (v/v) for 10 min at RT. The solution was dried undernitrogen flow. The residue was incubated for 30 min at RT in 1/1 TFA/DCM(v/v) and solvent was removed by a nitrogen flow. Product 61 waspurified by RP-HPLC.

MS (MW calculated) 61: 6445 g/mol (6445 g/mol)

1.8 ml of a solution of compound 61 (200 μM) in 1/1 (v/v)water/acetonitrile were mixed with 180 μl of a 1.5 mM solution ofmacrocyclic structure 34 in 1/1 (v/v) water/acetonitrile and dilutedwith 80/20 (v/v) water/acetonitrile to a total volume of 24 ml.Subsequently, the pH, was adjusted to 7.5 by addition of 0.5 M phosphatebuffer (pH 8.0). After incubating for 15 min, 62 was purified by RP-HPLCand lyophilized.

Detritylation of compound 62 was afforded by incubation for 30 ml with2/58/40 (v/v/v) TES/TFA/DCM. A two-step purification procedure employingRP-HPLC and SEC (Superdex 200, flow rate: 0.75 ml/min) yielded product63.

MS (MW calculated) 63: 15518 g/mol (15526 g/mol)

Structural element R represents the succinimidyl-containing product ofthe Michael addition of the neighboring proteophore thiol to themaleimido group of compound 37 or N-ethyl-maleimide, respectively.

Aliquots (9 nmol) of compound 63 were incubated with 15 eq ofN-ethyl-maleimide or 37, respectively, for 3 min in 70 μl 1/4 (v/v)acetonitrile/phosphate buffer 100 mM (pH 7.5). Subsequent purificationby SEC (Superdex 200, flow rate: 0.75 ml/min) gave products 64 or 65,respectively. Compound Retention time 64 20.2 min 65 13.3 minIX-4) Release of Insulin from Insulin MPIC 64 and 65

Release of insulin from Insulin MPIC 64 or 65, respectively, waseffected by linker hydrolysis in aqueous buffer. A ca. 10 μM solution ofconjugate was incubated in 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 3mM EDTA and 0.005% Tween. Samples were taken at time intervals andanalyzed by HPLC and UV detection. The peak correlating with theretention time of native insulin was integrated and plotted againstreaction time, and curve-fitting software was applied to estimate thecorresponding halftime of release. Under these conditions, insulin wasreleased from 64 with a halftime of approximately 150 min and fromcompound 65 with a halftime of ca 230 min.X—Synthesis of Reference Insulin Conjugates 68-75

150 μl of a solution of 0.64 M DIC, 0.56 M 8 and 0.29 M N-hydroxysuccinimide in DMF were prepared and incubated for 30 min. 18 mg insulinwere dissolved in 1.6 ml 20/30/50 (v/v/v) DMSO/DMF/water and 50 μl DIEAwere added, followed by addition of 50 μl of the preincubated cocktailcontaining compound 8. After incubation for 30 min another 100 μl ofpreactivated 8 were added. After incubation for 30 min product 66 waspurified by RP-HPLC. Acetonitrile was removed in vacuo and cleavage ofthe Mmt-protecting group was afforded by addition of 98/2 (v/v) TFA/TESuntil an intensive yellow colour was observed. Product 67 was obtainedby RP-HPLC purification.

MS (MW calculated) 67. 5893 g/mol (5896 g/mol)

Structural element R represents the succinimidyl-containing product ofthe Michael addition of a thiol to the maleimido group of compound 37,N-ethyl-maleimide, Maleimide-PEG5k, Maleimide-PEG20k orMaleimide-PEG2x20k, respectively. Thiols are associated with themodified insulins 54 or 67, respectively.

71 μl of a solution of 67 (464 μM) in 1/4 (v/v) acetonitrile/water wereincubated with 3.3 μl of N-ethyl maleimide (100 mM, 5 eq) in 1/1 (v/v)acetonitrile/water and 10 μl 0.5 M phosphate buffer (H 8.0) for 3 min.Purification of compound 68 was afforded by SEC (Superdex 200).

71 μl of a solution of 67 (464 μM) in 1/4 (v/v) acetonitrile/water weremixed with 7 μl of a solution of 10 mM Maleimide-PEG5k (1 eq) in 1/4(v/v) acetonitrile/water and 10 μl of 0.5 M phosphate buffer (pH 8.0)and incubated for 15 min. Compound 69 was purified by SEC (Superdex200).

Compounds 70 and 71 were obtained according to the procedure used forthe conjugation of 69 to 37 or Maleimide-PEG20k, respectively.

43 μl of a solution of 67 (464 μM) in 1/4 (v/v) acetonitrile/water weremixed with 22 μl of a solution of 2 mM Maleimide-PEG2x20k (2 eq) in 1/4(v/v) acetonitrile/water and 10 μl of 0.5 M phosphate buffer (pH 8.0)and incubated for 15 min. Compound 72 was purified by SEC (Superdex200).

11.4 μl of a solution of 54 (2.9 mM) in 1/4 (v/v) acetonitrile/waterwere mixed with 50 _82 l 1/1 (v/v) acetonitrile/water, 7 μl of a 100 mMsolution of N-ethyl maleimide (10 eq) and 10μl of 0.5 M phosphate buffer(pH 8.0) and incubated for 3 min. Compound 73 was purified by SEC(Superdex 200).

11.4 μl of a solution of 54 (2.9 mM) in 1/4 (v/v) acetonitrile/waterwere mixed with 50 μl 1/1 (v/v) acetonitrile/water, 15 μl of a 10 mMsolution of Maleimide-PEG5k (2eq) in 1/4 (v/v) acetonitrile/water and10μl of 0.5 M phosphate buffer (pH 8.0) and incubated for 15 min.Compound 74 was purified by SEC (Superdex 200, flow rate: 0.75 ml/min).Compound 75 was obtained accordingly through reaction of 74 withMaleimide-PEG20k.

Preparative SEC of compounds 68-75 yielded fractions of 1.4-2.2 ml.Concentrations were determined by measuring UV extinction at 275 nm,assuming an average coefficient of extinction of ε₂₇₅=7500. CompoundE₂₇₅/mOD Concentration/μMol Retention time/min 68 97 12.9 23.0 69 10213.6 18.8 70 33 4.4 16.1 71 60 8.0 15.9 72 38 5.0 14.3 73 100 13.3 23.374 75 10.0 17.8 75 59 7.9 14.1XI) Synthesis of Insulin DPICs

76 was obtained according to the standard solid-phase synthesisprotocol. The amino acid sequence Fmoc-Dpr(ivDde)-OH, Fmoc-Lys(Fmoc)-OH,Fmoc-Ado-OH, Fmoc-Lys(Fmoc)-OH, Fmoc-Ado-OH and Boc-,βAla-OH werecoupled to Sieber amide resin.

Resin was treated with DMF/hydrazine 98/2 (v/v), washed and agitatedwith 5 eq maleimidopropionic acid and 5 eq DIC in DMF for 30 min.Product was cleaved from resin with TFA/TES/water 95/3/2 (v/v/v). Afterevaporation of solvent, the residue was taken up in 3/1 (v/v)DMF/collidine and reacted for 30 min with a solution of 15 eq 8preactivated for 15 min with 10 eq DIC in DMF. After acidification withacetic acid, product 77 was purified by RP-HPLC.

MS (MW calculated) 77: 3233,7 g/mol (3236,0 g/mol).

Structural element 77′ represents the succinimidyl-containing product ofthe Michael addition of a thiol group to the maleimido group of compound77 after Mmt protecting group removal. Thiol groups are eitherassociated with the insulin-linker conjugate or with a dendron-insulinconjugate.

XI-2-1) Synthesis of 1st Generation Insulin-N^(εB29)-Mpa-dendrimer (78)

2.9 mg 67 were dissolved in 100 μl 4/1 acetonitrile/water and mixed with180 μl of a solution of 77 (3.4 mM) in 4/1 acetonitrile/water. 20 μl of0.5 M phosphate buffer (pH 7.5) were added and the solution was treatedfor 30 min with in an ultrasonic bath. The cocktail was mixed with 300μl TPA and 10 μl TES agitated for 30 s. After dilution with 1/1acetonitrile/water, compound 78 was purified by RP-HPLC. Yield: 43%.

MS (MW calculated) 78: 8035 g/mol (8042 g/mol).

XI-2-2) Synthesis of 2nd Generation Insulin-N^(εB29)-Mpa-dendrimer (79)

350 μl of a solution of 78 (420 μM) in 4/1 acetonitrile/water were mixedwith 150 μl of a 5.2 mM solution of dendron 77 in 4/1acetonitrile/water. 20 μl of 0.5 M phosphate buffer (pH 7.5) were addedand the solution was treated for 30 min in an ultrasonic bath. Thecocktail was mixed with 500 μl TFA and 20 μl TES and agitated for 30 s.After dilution with 1/1 acetonitrile/water, compound 79 was purified byRP-HPLC.

Yield: 40%

MS (MW calculated) 79: 16621 g/mol (16622 g/mol).

XI-2-3) Synthesis of 3rd Generation Insulin-N^(εB29)-Mpa-dendrimer (80)

46 μl of a solution of 79 (235 μM) in 4/1 acetonitrile/water were mixedwith 60 μl of a 5.2 mM solution of dendron 77 in 4/1 acetonitrile/water.20 μl of 0.5 M phosphate buffer (pH 7.5) were added and the solution wastreated for 30 min in an ultrasonic bath. The cocktail was mixed with100 μl TFA and 5 μl TES and agitated for 30 s. After dilution with 1/1water/acetonitrile compound 80 was purified by HPLC.

Yield: 38%

MS (MW calculated) 80: 50967 g/mol (50967 g/mol)X-3) Synthesis of End-Capped Dendrimers 81, 82, 83 and 84 Based on 2ndGeneration Insulin-N^(εB29)-Mpa-dendrimer

Structural element R represents the succinimidyl-containing product ofthe Michael addition of a thiol group of 77′ to the maleimido group ofcompound 37, 38, N-ethyl-maleimide, or Maleimide-PEG5k, respectively.

XI-3-1) Synthesis of 81

70 μl of a solution of 79 (230 μM) in 1/1 acetonitrile/water were mixedwith 10 μl of a 24 mM solution of N-ethyl-maleimide in acetonitrile. pHwas adjusted to 7.5 with 0.5 M phosphate buffer (pH 7.5) and thesolution was incubated for 30 min at RT. Purification by SEC (Superdex200, flow rate: 0.75 ml/min) gave compound 81.

SEC (retention time) 81: 20.28 min

MS (MW calculated) 81: 18629 g/mol (18624 g/mol)

XI-3-2) Synthesis of 82

70 μl of a solution of 79 (230 μM) in 1/1 acetonitrile/water were mixedwith 3 mg 38. The pH was adjusted to 7.5 with 0.5 M phosphate buffer (pH7.5) and the solution was incubated for 30 min at RT. Purification bySEC (Superdex 200, flow rate: 0.75 ml/min) yielded compound 82.

SEC (retention time) 82: 14.5 min

XI-3-3) Synthesis of 83

70 μl of a solution of 79 (230 μM) in 1/1 acetonitrile/water were mixedwith 2.6 mg Maleimide-PEG5k. The pH was adjusted to 7.5 with 0.5 Mphosphate buffer (pH 7.5) and the solution was incubated for 30 min atRT. Purification by SEC (Superdex 200, flow rate: 0.75 ml/min) gavecompound 83.

SEC (retention time) 83: 12.9 min

XI-3-4) Synthesis of 84

115 μt of a solution of 79 (65 μM) in 1/1 acetonitrile/water were mixedwith 5 mg 37. The pH was adjusted to 7.5 with 0.5 M phosphate buffer (pH7.5) and the solution was incubated for 30 min at RT. Purification bySEC (Superdex 200, flow rate: 0.75 ml/min) gave compound 84.

SEC (retention time) 84: 10.9 min

Thiol groups are either associated with the insulin-linker conjugate orwith a dendron-insulin conjugate.

Insulin in DMSO was mixed with a solution of 0.9 eq 11 in DMSO. Theresulting solution was adjusted to pH 8.0 with DIEA and stirred for 1.5h at RT. RP-HPLC purification gave Mmt-protected intermediate 85.

MS (MW calculated) 85: 6350 g/mol (6350 g/mol)

Regioselectivity of the monoconjugation was determined by reduction of85 with DTT (10 mM) in 0.5 M phosphate buffer pH 8.0 for 1 h at RT andsubsequent analysis of the insulin A- and B-chains by LC-MS.

After lyophilization, compound 85 was mixed with 95:5 (v/v)TFA/triethylsilane and stirred for 5 min. Volatiles were removed undernitrogen flow and 86 was purified by RP-HPLC and lyophilized.

MS (MW calculated) 86: 6077 g/mol (6077 g/mol)

XI-4-2) Synthesis of 1st GenerationN^(αA1)-thiollinker-insulin-dendrimer 87

Synthesis of compound 87 was performed in analogy to the synthesis ofcompound 78.

Yield: 27%

MS (MW calculated) 87: 8218 g/mol (8223 g/mol).

XI-4-3) Synthesis of 2nd GenerationN^(αA1)-thiollinker-insulin-dendrimer 88

Synthesis of compound 88 was performed in analogy to the synthesis ofcompound 79.

Yield: 17%

MS (MW calculated) 88: 16809 g/mol (16809 g/mol).

XI-4-4) Synthesis of 3rd GenerationN^(αA1)-thiollinker-insulin-dendrimer 89

Synthesis of compound 89 was performed in analogy to the synthesis ofcompound 80.

Yield: 44%

MS (MW calculated) 89: 51117 g/mol (51155 g/mol).XI-5) Synthesis of End-Capped Dendrimers 90 and 91 Based on 1st or 2ndGeneration N^(αA1)-Thiollinker-Insulin-Dendrimers

Structural element 37′represents the succinimidyl-containing product ofthe Michael addition of a thiol group to the maleimido group of compound37. Thiol groups are associated with a generation 1 dendron-insulinconjugate.

50 μl of a solution of 87 (450 μM) in 1/1 acetonitrile/water were mixedwith 3.5 mg of 37. The pH was adjusted to 7.5 with 0.5 M phosphatebuffer (pH 7.5) and the solution was incubated for 30 min at RT.Purification by SEC (Superdex 200, flow rate 0.75 ml/min) gave compound90.

SEC (retention time ) 90: 13.0 min

Structural element 37′ represents the succinimidyl-containing product ofthe Michael addition of a thiol group to the maleimido group of compound37. Thiol groups are associated with a generation 2 dendron-insulinconjugate.

75 μl of a solution of 88 (206 μM) in 1/1 acetonitrile/water were mixedwith 9 mg of 37. The pH of the solution was adjusted to 7.5 with 0.5 Mphosphate buffer (pH 7.5) and the solution was incubated for 30 min atRT. Purification by SEC (Superdex 200, flow rate 0.75 ml/min) yieldedcompound 91.

SEC (retention time) 91: 10.9 min

XI-6@) Release of Insulin from Insulin DPIC 90 and 91

SEC-eluates of 90 and 91 were incubated with 10 mM HEPES buffer (pH7.4), 150 mM NaCl, 3 mM EDTA and 0.005% Tween at 37° C., and the amountof free insulin was quantified by HPLC using UV detection at 215 nm(FIG. 5).

XII—Analysis of Efficiency of Encapsulation

Analysis was performed on a BIAcore 2000 surface plasmon resonanceinstrument using CM5 sensor chips (Biacore).

XII-1) Preparation of Sensor Chips

A sensor chip was mounted to the instrument and RAMFc (rabbit anti mouseFc antibody, BIAcore) was immobilized according to Karlsson et al. (J.Immun. Meth, 200, 1997, 121-133) using EDC/NHS activation. Capping ofactivated, unreacted surface carboxy groups was effected byethanolamine.

XII-2) Interaction Analysis between Proteophor-Encapsulated Insulin andAnti-Insulin Antibodies

Insulin and insulin conjugates 68-75, 57-59 and 81-84 were subject toanalysis.

For sample application, dissociation and regeneration, standard flowbuffer was used containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA,0,005% Tween 20.

Three murine anti insulin monoclonal antibodies (Advanced ImmunoChemicalInc., clones C7C9, 8E2, or 7F8, respectively,) were loaded ontoprespecified sensor chip areas by injection of 15 μl of a solution of 30μg/ml. A fourth sensor area was used for reference purposes. Afterequilibration for 2.5 min, 150 μl of a 100 nM solution of insulin orinsulin conjugate were injected and flowed across all four sensor areas.A dissociation phase of 3 min was followed by removal of theanti-insulins by injection of 60 μl glycine buffer (pH 2.0) andregeneration of the sensor chip surface. Encapsulation efficiency wasmeasured by recording the refractive index units (RU) of each of thefour, sensor areas before dissociation.

Highly efficient encapsulation of insulin was achieved by conjugatingthe protein to proteophore structures 84, 58 or 59, respectively, asevidenced by suppression of antibody binding.

While there have been described what are presently believed to be thepreferred embodiments of the invention, those skilled in the art willrealize that changes and modifications may be made without departingfrom the spirit of the invention. It is intended to claim all suchchanges and modifications as fall within the true scope of theinvention.

ABBREVIATIONS

-   Boc t-butyloxycarbonyl-   DBU 1,3-diazabicyclo[5.4.0]andecene-   DCM dichloromethane-   (iv)Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)3-methyl-butyl-   DIC diisopropylcarbodiimide-   DIEA diisopropylethylamine-   DMAP dimethylamino-pyridine-   DMF N,N-dimethylformamide-   DMSO dimethylsulfoxide-   Dpr diaminopropionic acid-   DTT dithiothreitol-   EDC-HCl l-Ethyl-3-(3′-dimethylaminopropyl) carbodiimide    hydrochloride-   Endo-GluC endoproteinase-GluC-   eq stoichiometric equivalent-   Fmoc 9-fluorenylmethoxycarbonyl-   Fmoc-PP—OH Fmoc-aminoethyl-undecaethyleneoxide-propionic acid-   Fmoc-Ado-OH Fmoc-8-amino-3,6-dioxaoctanoic acid-   Hb hemoglobin (human)-   HEPES N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid)-   HOBT N-hydroxybenzotriazole-   LCMS mass spectrometry-coupled liquid chromatography-   Mal maleimidopropionyl-   Mmt 4-methoxytrityl-   Mpa mercaptopropionyl-   MS mass spectrum-   MSNT 1-(mesitylen-2-sulfonyl)-3-nitro-1H-1,2,4-triazole-   Mtt 4-methyltrityl-   MW molecular mass-   NHS N-hydroxy succinimide-   RP-HPLC reversed-phase high pressure liquid chromatography-   RT room temperature-   RU response units-   SEC size exclusion chromatography-   SPDP succinimidyl 3-(2-pyridyldithio) propionate-   S-tBu t-butylthio-   Suc succinimidopropionyl-   TBTU O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    tetrafluoroborate-   TCEP tricarboxyethylphosphine-   TES triethylsilane-   TFA trifluoroacetic acid-   THF tetrahydrofurane-   Trt trityl, triphenylmethyl-   UV ultraviolet

1-31. (canceled)
 32. A composition comprising: a hyperbranched polymerattached to a core; and a biologically active moiety; whereby thebiologically active moiety is attached to the core by means of asubstantially non-enzymatically cleavable linker L.
 33. The compositionof claim 32, wherein the hyperbranched polymer is water soluble.
 34. Thecomposition of claim 32, wherein the hyperbranched polymer contains atleast two molecular chains, which molecular chains are of sufficientlength to be so arranged as to form a cavity to accommodate thebiologically active moiety.
 35. The composition according to claim 32,wherein the polymer chains contain linear, branched or cyclical alkylchains.
 36. The composition according to claim 32, wherein furthergroups are present in the polymer chains, the further groups beingselected from the groups consisting of S, N, O, (—S—S)—, oxyethylene,oxypropylene and oxybutylene, amide-C(O)NH— or C(O)NR—, —S-succinimido,amino (—NR—), carboxylic ester (—C(O)O—), sulfonamide (—S(O)₂—NR—),carbamate (—O—C(O)—NR—), carbonate (—OC(O)—O—), sulfone (—S(O)₂—), ether(—O—), oxime (—CR═N-O—), hydrazone (—CR═N—), NR—), urea (—NR—C(O)—NR—),thiourea (—NR—C(S)—NR—), carbohydrate, glyceryl, phosphate(—O—P(O)(OR)O—), phosphonate (—P(O)(OR)O—), saturated and nonsaturated(hetero)cyclic groups, in which R is H, a linear, branched or cyclicalalkyl groups which may contain further functional groups or heteroatoms.
 37. The composition according to claim 32, wherein the molecularchains contain sterically demanding capping groups C.
 38. Thecomposition according to claim 37, wherein the capping groups C withinthe molecular chains contain linear, branched or cyclical alkyl chains.39. The composition according to claim 37, wherein the capping groups Ccontain further groups selected from the groups consisting of S, N, O,(—S—S)—, oxyethylene, oxypropylene and oxybutylene, amide —C(O)NH— orC(O)NR—, —S-succinimido, amino (—NR—) carboxylic ester (—C(O)O—),sulfonamide (—S(O)₂—NR—), carbamate (—O—C(O)—NR—), carbonate(—O—C(O)—O—), sulfone (—S(O)₂—), ether (—O—), oxime (—CR═N—O—),hydrazone (—CR═N—NR—), urea (—NR—C(O)—NR—), thiourea (—NR—C(S)—NR—),carbohydrate, glyceryl, phosphate (—O—P(O)(OR)O—), phosphonate(—P(O)(OR)O—), saturated and nonsaturated (hetero)cyclic groups in whichR is H, linear, branched or cyclical alkyl groups which may containfurther functional groups or hetero atoms.
 40. The composition accordingto claim 37, wherein the capping groups C are highly branched moleculescontaining centers with a branching degree of between 2 and
 6. 41. Thecomposition of claim 40, wherein the capping groups C include at leastone thio-succinimido moiety resulting from a reaction between amaleimido group and a thiol group.
 42. The composition according toclaim 32, wherein the biologically active moiety is a biopolymer. 43.The composition according to claim 32, wherein the biologically activemoiety is selected from the group of protein or polypeptides consistingof ACTH, adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin(AAT), alfa-1 alfa-1 proteinase inhibitor (API), alteplase,anistreplase, ancrod serine protease, antibodies (monoclonal orpolyclonal, and fragments or fusions), antithrombin III, antitrypsins,aprotinin, asparaginases, biphalin, bone-morphogenic proteins,calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide,enkephalins, erythropoietins, factor VIIa, factor VIII, factor VIIIa,factor IX, fibrinolysin, fusion proteins, follicle-stimulating hormones,granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon,glucocerebrosidase, granulocyte macrophage colony stimulating factor(GM-C′SF), phospholipase-activating protein (PLAP), gonadotropinchorionic (hCG), hemoglobins, hepatitis B vaccines, hirudin,hyaluronidases, idurnonidase, immune globulins, influenza vaccines,interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-1 receptorantagonist (rhIL-1ra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c,beta 1 a, beta 1 b, gamma 1 a, gamma 1 b), keratinocyte growth factor(KGF), transforming growth factors, lactase, leuprolide, levothyroxine,luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase,papain, parathyroid hormone, PDGF, pepsin, platelet activating factoracetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin,sermorelin, superoxide dismutase (SOD), somatropins (growth hormone),somatostatin, streptokinase, sucrase, tetanus toxin fragment, tilactase,thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumornecrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator(tPA), TSH, urate oxidase, urokinase, vaccines, and plant protein suchas lectins and ricins.
 44. The composition of claim 32, wherein thebiologically active moiety is insulin.
 45. The composition according toclaim 32, wherein the biologically active moiety is an organic smallmolecule bioactive agent.
 46. The composition according to claim 45,wherein the biologically active moiety is selected from the group oforganic small molecule bioactive agents consisting of central nervoussystem-active agents, anti-infective, anti-neoplastic, antibacterial,anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal,vasodilating, vasoconstricting, and cardiovascular agents.
 47. Thecomposition of claim 32, wherein the biologically active moiety is ananti-sense or interfering oligonucleotide.
 48. The composition accordingto claim 32, wherein the encapsulating organic compound has a dendriticstructure.
 49. The composition of claim 32, wherein the hyperbranchedmolecule comprises a first branching unit B with a first branchingcenter Bc at least two first branching functional groups Bfg and atleast two molecular chains connected to the at least two first branchingfunctional groups Bfg
 50. The composition of claim 49, wherein the firstbranching center Bc contains groups selectedfrom >C<, >CH—, >CR—, >N—, >P— which are linkable linked to the firstbranching functional groups Bfg.
 51. The composition of claim 49,wherein the first branching unit B contains linear, branched or cyclicalalkyl chains.
 52. The composition of claim 50, wherein the firstbranching unit B further comprises groups selected from the groupsconsisting of S, N, O, (—S—S), oxyethylene, oxypropylene andoxybutylene, amide —C(O)NH— or (C(O)NR—, —S-succinimido, amino (—NR—),carboxylic ester (—C—(O)O—), sulfonamide (—S(O)₂—NR—, carbamate(—O—C(O)—NR—), carbonate (—O—C(O)—O—), sulfone (—S(O)₂—), ether (—O—),oxime (—CR═N—O), hydrazone (—CR═N—NR—), urea (—NR—C(O)—NR—), thiourea(—NR—C(S)—NR—), carbohydrate, glyceryl, phosphate (—O—P(O)(OR)O—),phosphonate (—P(O)(OR)O—), saturated and nonsaturated (hetero)cycliccompounds, in which R is H or a linear, branched or cyclical alkylgroups which may contain further functional groups or hetero atoms. 53.The composition of claim 48, wherein the first branching functionalgroups Bfg are selected from amino (—NRH), carboxylic acid (—C(O)OH) andderivatives, sulfonic acid (—S(O)₂—OH) and derivatives, carbonate(—O—C(O)—O—) and derivatives, hydroxyl (—OH), aldehyde (—CHO), ketone(—CRO), hydrazine (H₂ N—NR—), isocyanate (—NCO), isothiocyanate (—NC S),phosphoric acid (—O—P(O)(OR)(OH) and derivatives, phosphonic acid,(—P(O)(OR)OH) and derivatives, haloacetyl, alkyl halides, maleimide,acryloyl, arylating agents like aryl fluorides, hydroxylamine,disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone,diazoalkanes, diazoacetyl compounds, epoxide, oxirane, aziridine, inwhich R is H or a linear, branched or cyclical alkyl group which maycontain further functional or hetero atoms or acryl groups.
 54. Thecomposition of claim 32, wherein the cleavable linker L can be cleavedby TCEP, TFA, DTT, or buffer.
 55. The composition of claim 32, whereinthe cleavable linker L further contains linker functional groups adaptedto react between the cleavable linker L and the biologically activemoiety by the formation of a chemical bond.
 56. The composition of claim55, wherein the linker functional groups are selected from the groupsconsisting of amino (—NRH), carboxylic acid (—C(O)OH) and derivatives,sulfonic acid (—S(O)₂—OH) an derivatives, carbonate (—O—C(O)—O) andderivatives, hydroxyl (—OH), aldehyde (—CHO), ketone (—CRO), isocyanate(—NCO), isothiocycanate (—NCS), haloacetyl, alkyl halides, maleimide,acryloyl, arylating, agents, aryl fluorides, disulfides, pyridyldisulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetylcompounds, epoxide, oxirane, aziridine, in which R is H or a linear,branched or cyclical alkyl group which may contain further functionalgroups or hetero atoms or aryl groups.
 57. The composition of claim 32,wherein the cleavable linker L is a traceless prodrug linker andcontains a hydrolysable ester bond which can be hydrolysed and acarbamate.
 58. The composition of claim 57, wherein the hydrolysableester bond is a phenol ester.
 59. The composition of claim 49, whereinthe composition includes a second branching unit B′ with a secondbranching centre Bc′ at least two second branching functional groupsBfg′ wherein at least one of the at least two molecular chains isconnected between one of the at least two first branching functionalgroups Bfg and one of the at least two second branching functionalgroups Bfg′.
 60. The composition of claim 58 further including a secondcleavable linker L′ comprising at least one second functional group Lfg′which is connectable with the biologically active moiety.
 61. A methodfor selectively delivering a biologically active moiety to a target,which method comprises providing the composition according to claim 32;bringing the composition into contact with a liquid containing thetarget.
 62. A drug containing the composition of claim 32.